INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Atopy is the principal causative factor of asthma and it is believed that as many as 10% of children suffer from asthma as a result of atopy (1). Of further concern is the fact that both the prevalence and the severity of the disease are increasing worldwide, especially in the developed countries (2). Atopy is characterized by exaggerated T helper cell type 2 (Th2) lymphocyte responses to common allergens with sustained, enhanced production of allergen-specific IgE (3). Atopic asthma, which is characterized by increased bronchial responsiveness to methacholine or histamine (1), arises as a result of complex interactions between several genetic and environmental factors.

Previous studies have found linkage of atopy and bronchial hyperresponsiveness to markers on chromosomes 11q13 (4, 5). The beta chain of the high-affinity receptor for IgE (Fc RI-) has been identified as the most likely candidate for this linkage (6). Two coding variants in exon 6, I181L (a substitution of leucine for isoleucine at amino acid position 181) and V183L (a substitution of leucine for valine at amino acid position 183) in the fourth transmembrane domain of Fc RI- have been previously described in 13% of British subjects (7). I181L showed a significant association with measures of atopy in the random patient sample and was shown to be maternally inherited in a group of 60 unrelated nuclear families with allergic asthmatic probands (7). V183L was found in only one patient, who also had the I181L variant (7).

A new coding polymorphism in exon 7 of Fc RI-, which changes amino acid residue 237 from glutamic acid to glycine (E237G) in the cytoplasmic tail of the protein, has been described in 5.3% of an Australian general population sample (8). E237G-positive subjects had a significantly elevated skin test response to grass and house dust mite, RAST to grass, and bronchial reactivity to methacholine, while the relative risk of E237G-positive subjects having asthma compared with those individuals without the polymorphism was 2.3 (8). Of further interest was the finding that E237G showed a much stronger association with bronchial hyperresponsiveness than with atopy (8), and that this genetic linkage of Fc RI- to bronchial hyperresponsiveness, independent of atopy, has also been reported in Australian asthmatic sib pairs (9, 10).

The role of the -chain is rather obscure, although expression studies suggest that it might play a role in signal transduction and in autophosphorylation of the receptor (11). In addition, Fc RI- has been shown to associate with the low-affinity IgG receptor (FcRIII) in macrophages and in mast cells (12). The functional significance of the I181L and E237G mutations has not been elucidated. Tyrosine phosphorylation of clustered FC RI subunits occurs in a 19-amino acid motif in the cytoplasmic tails of the - and -chains of the receptor, referred to as an "antigen receptor activation motif" (ARAM) (12), and it has been suggested that I181L could influence signaling by the cytoplasmic tails of the protein by exerting a pleiotropic effect on the ARAM-mediated activation or by making the receptor more sensitive to ligand (13). E237G introduces a hydrophobicity change in the C-terminus of Fc RI- and could affect the intracellular signaling capacity of Fc RI as this mutation is adjacent to the ARAM (8).

The present study was undertaken in order to document the prevalence of the I181L, V1831, and E237G variants of Fc RI- in a sample of black and white atopic asthmatics and control subjects in South Africa in an attempt to determine whether these variants contribute to the enhanced IgE responses in these groups, and also to determine whether the discrepancy in the prevalence of atopy in these groups (whites tend to be more atopic than blacks) could be attributed to these variants.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Forty-eight black asthmatics (25 women), 45 white asthmatics (22 women), 44 black control subjects (20 women), and 51 white control subjects (23 women) were screened for the I181L variant in exon 6 of the Fc RI- gene. Forty-one black (28 women) and 39 white (21 women) asthmatics, and 27 black (12 women) and 30 white (15 women) control subjects were screened for the V183L variant in exon 6 of the Fc RI- gene. Forty-one black asthmatics (22 women), 46 white (26 women) asthmatics, 42 black control subjects (19 women), and 51 white control subjects (17 women) were screened for the E237G mutation within the seventh exon of the -chain gene.

The black and white asthmatics were recruited from the Asthma and Respiratory Clinics of Hillbrow and Johannesburg Hospitals. The control subjects were either asymptomatic individuals who presented themselves as blood donors at the South African Blood Transfusion Services in Johannesburg, or outpatients from the Johannesburg Hospital Paediatric Nephrology Clinic. None of the control subjects had a history of atopy or any disease process associated with the respiratory system.

All individuals who participated in the study were 6 to 45 yr of age; of these the following were < 16 yr: 21 (9 girls) were white asthmatics, 16 (7 girls) were black asthmatics, 27 (11 girls) were white control subjects, 29 (15 girls) were black control subjects. Each subject gave informed consent (in the case of minors, consent was obtained from parents) to participate in the study, which was passed by the Committee for Research on Human Subjects of the University of the Witwatersrand.

Asthma and Atopy

Each patient had a history of bronchial asthma as defined by the American Thoracic Society, namely a history of transitory or prolonged episodes of dyspnea, coughing, and wheezing, but was otherwise healthy (14). The diagnosis was confirmed in each patient by the finding of at least one of the following: a forced expiratory volume in one second (FEV₁) < 70% of the predicted value, with reversibility of 20% or more after inhalation of a ₂-adrenergic bronchodilator, or a fall in peak expiratory flow rate of at least 20% during a standardized exercise test.

Atopy was defined as a positive skin prick test at least 2 mm greater than a negative control and a positive specific IgE titer (> 0.35 kU/L) or a high concentration of total serum IgE greater than published normal values for children, or greater than 400 kU/L in adults.

DNA Extraction and Amplification

Ten milliliters of venous blood was collected from all subjects into ethylenediaminetetraacetic acid (EDTA)-coated tubes and DNA was extracted from the blood according to the Higuchi method (15).

The prevalence of the I181L and V183L variants within the sixth exon of the Fc RI- gene were determined by means of amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The following primers, based on the published sequence of Fc RI- (16), were designed: IgE1: 5'-GTA TGT GTC ACT TTA AAA GGA CTG GTC-3'; IgE2: 5'-AAG AAA TGT TTC AGA AAA CCC AGG CCA-3'; IgE3: 5'-GGT GAG AAA CAG CAT CAT CAC TAC AAT-3'; IgE4: 5'-TCA TTT GTT GCT GTT CAA TAG GAA TTG-3'; IgE5: 5'-GTT GCT GTT CAA TAG GAA ATT GTA TTC-3'. Primers IgE1 and IgE2 amplify an internal control band of 323 bp; IgE1 and IgE3 amplify a wild-type band of 159 bp in the presence of isoleucine (ATT) at amino acid position 181; IgE4 and IgE2 amplify a mutant band of 214 bp in the presence of the leucine (TTG) substitution at amino acid position 181; and primers IgE5 and IgE2 amplify a mutant band of 209 bp in the presence of the valine (GTG TTG) to leucine substitution at amino acid position 183. Briefly, the target sequence was amplified in a 50 µl reaction volume containing 0.4 µg genomic DNA, 0.2 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), 10 mM Tris-HCl buffer (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂ (0.01% gelatine), 6.7 µM IgE1, 3.4 µM each of IgE2 and IgE3, and 5.8 µM IgE4 or 6.0 µM IgE5 amplification primers. Negative PCR controls contained all the reagents, except genomic DNA. The reaction mixture containing all components except enzyme was overlaid with 50 µl mineral oil and heated to 95° C for 5 min in a Hybaid DNA thermal cycler (Hybaid, London, UK). After a cooling step which reduced the sample temperature to 60° C, 1 unit of Taq DNA polymerase was added. The samples were then processed through 35 cycles of 94° C denaturation for 1 min, 60° C annealing for 2 min, and extension at 72° C for 2 min. The procedure was completed by the continuation of the last 72° C incubation for a further 10 min. Electrophoresis of the ARMS products was carried out in 4% (wt/vol) agarose gels (3:1 agarose: low melting point agarose) containing ethidium bromide and visualized under ultraviolet illumination.

The prevalence of the E237G variant was determined using the ARMS-PCR primers and protocol described by Hill and Cookson (8).

In order to confirm the mutations in exons VI and VII, direct sequencing with thermo sequenase and ³³P-labeled terminators (Amersham Life Science, Inc., Cleveland, OH) was used. The following sequencing primers were designed: 5'-GTATGTGTCACTTTAAAAGGAGTGGTC-3' for exon VI and 5'-AAGAAATGTTTCAGAAAACCCCAGGCCA-3' for exon VII.

Statistical Analyses of Data

The comparison of the prevalence of the I181L and E237G variants in black and white asthmatic and control groups was done by means of the two-tailed Chi-square test with a Yates correction for continuity. The prevalence of the V183L variant in the black and white asthmatic and control groups was too low to warrant statistical analysis.

Materials

Agarose, deoxyribonucleoside triphosphates (dNTPs), and Taq DNA polymerase were obtained from Promega (Madison, WI). The oligonucleotide primers used to detect the I181L and V183L variants were synthesized by Perkin Elmer-Applied BioSystems (Cheshire, UK); the oligonucleotide primers that were used for the detection of the E237G variant and the sequencing primers, were synthesized by Genosys Biotechnologies Inc. (Woodlands, TX). The DNA molecular weight markers were supplied by BioVentures Inc. (Murfreesboro, TN). All other reagents used were of analytical grade (Merck, Darmstadt, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Prevalence of I181L in Black and White Asthmatic and Control Subjects

Figure 1 shows the results of an ARMS test for the I181L in a group of asthmatic and control individuals. The prevalence of the I181L variant of Fc RI- in black and white asthmatics and in black and white control subjects is shown in Table 1. All the asthmatics and controls were found to be heterozygous by ARMS for I181L (i.e., all individuals had both the wild-type isoleucine and the mutated leucine at amino acid position 181). There was a significant difference in the prevalence of I181L between white asthmatics (28%) and white control subjects (3%) (p = 0.00001) and between black control subjects (16%) and white control subjects (p = 0.002). There was no difference in the frequency of I181L between black asthmatics and black control subjects. The black and white asthmatic and control individuals who were positive for I181L were all heterozygotes.

View larger version (40K): [in this window] [in a new window]   Figure 1.   Results of an ARMS test for I181L. Lane 1, molecular weight markers; lanes 2 and 4, negative ARMS-PCR controls; lanes 5, 7, 10, and 12, asthmatics who were heterozygous for I181L; lanes 3, 6, 9, and 11, individuals who were negative for I181L.

V183L was only found in one black asthmatic, who also had the I181L and E237G mutations. V183L was not found in any of the other groups tested.

Figure 2 shows the DNA sequence of the PCR product of an asthmatic patient who showed the I181L mutation in one of the alleles by the ARMS technique. The mutation could not be detected by either using the specifically designed sequencing primer or by using primer IgE1. This was also the case for other individuals who were heterozygous for the I181L mutation. All sequences were the wild types.

View larger version (120K): [in this window] [in a new window]   Figure 2.   DNA sequence of exon 6 showing the wild type in a heterozygous asthmatic individual who showed the I181(ATT TTG)L mutation by ARMS-PCR.

Prevalence of E237G in Black and White Asthmatic and Control Subjects

Figure 3 shows the results of the ARMS test to detect E237G. Figures 4-6 show the DNA sequences of an individual homozygous for the E237G mutation, a heterozygous individual, and a control individual without the E237G mutation respectively. Table 2 shows the frequencies of E237G in black and white asthmatics and in black and white control subjects. The frequency of the E237G variant in black asthmatics was 20% as compared with 12% in white asthmatics, but this difference was not statistically significant. There was a higher prevalence of the mutation in black control subjects (20%) than in white control subjects (5%) (p = 0.002).

View larger version (35K): [in this window] [in a new window]   Figure 3.   Results of an ARMS test for E237G. Lane 1, molecular weight markers; lane 6, negative ARMS-PCR control; lanes 11 and 13, two black asthmatics who were homozygous for E237G; lanes 12 and 15, asthmatics who were heterozygous for E237G; lanes 2-5, 7-10, and 14, individuals who did not have the E237G mutation.

View larger version (102K): [in this window] [in a new window]   Figure 4.   DNA sequence of the E237G substitution showing the E(GAA GGA)G mutation in a homozygote.

View larger version (104K): [in this window] [in a new window]   Figure 5.   DNA sequence of the E237G substitution showing the E(GAA GGA)G mutation in one of the alleles and the E(GAA) sequence in the wild type allele in a heterozygous individual.

View larger version (119K): [in this window] [in a new window]   Figure 6.   DNA sequence of the E(GAA) wild type in a homozygote.

Of the asthmatics and control subjects tested, only two were homozygous for E237G, and both of these individuals were black asthmatics.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The finding of the strong association of I181L with atopy (7) may help to explain the apparent lack of atopy in the black population. Studies of communities in Gambia have shown that although the black communities have IgE levels that are significantly higher than those of adjacent white communities, much less atopic disease is seen in these ethnic communities (17). Furthermore, it has been shown previously that IgE levels are high in both normal and asthmatic groups in the black population (18, 19), highlighting the fact that high IgE levels in well-developed communities (in the present study, represented by the white population) are associated with atopic disease, whereas much higher IgE levels in underdeveloped communities (as represented by the black population in the present study) are not, and tend to be indications of helminthic and other parasitic infections. In the present study the I181L substitution was identified by ARMS-PCR. It was found that there was no difference in the prevalence of I181L between the black asthmatics and black control individuals, suggesting that the presence of I181L might not predispose to the development of atopy in this population. However, I181L had a significantly higher frequency in the white asthmatics as compared with the white control subjects, which suggests that in whites, the presence of I181L may predispose to atopy. This result supports the findings of Shirakawa and coworkers (7) and appears to support the hypothesis on the evolution of atopy from parasite defense systems which might explain the apparent lack of atopy in our black population. However, failure to show the I181L (ATT TTG) mutation on direct sequencing of the PCR products of heterozygotes confirms the work of other studies (20). Our results suggest that the mutations may be in a pseudogene.

A number of hypotheses have been put forward to explain why natural selection has allowed allergy to become so widespread in developed communities. The most common, and possibly the most plausible theory is that the allergic response evolved from a defense system initially intended to help the body cope with parasitic infections, and that individuals with a genetic make-up that enabled them to mount an effective immune response to helminths and other parasites would have a survival advantage over individuals who lacked such a defense system (21). In support of this hypothesis, epidemiologists have found that allergic disease is less common in developing than in developed nations where public health measures have practically eradicated exposure to parasites (21). Further support for this hypothesis comes from a study that showed that Fc RI is expressed on eosinophils in patients with hypereosinophilia, and that the receptor participates in a protective immune response against parasite infections in addition to its role in allergic manifestations, suggesting that Fc RI evolved to protect against pathogens rather than to induce allergy (22). And a recent study has demonstrated a bi-allelic RsaI polymorphism in Fc RI- to be associated with total serum IgE levels in Australian Aborigines endemically parasitized with helminths (23).

In the present study, E237G was found to be more prevalent in the black than in the white population. The fact that this mutation is more associated with bronchial hyperresponsiveness even in the absence of atopy (8) might explain its higher frequency in the black population which although not atopic, appears to have more severe asthma (as evidenced by the significantly higher asthma mortality rate) than their white counterparts (24). Although poor socioeconomic development has often been cited as a possible reason for the much higher asthma mortality rate seen in blacks (21, 24), the finding in the present study of a higher frequency in the black population of the E237G mutation which is associated with bronchial hyperresponsiveness (and therefore by implication with increased asthma severity) might point to the existence of a genetic locus on chromosome 11q13 that might influence asthma severity in this population. A similar scenario exists in the case of the ₂-adrenergic receptor gene. A Glycine¹⁶ Arginine mutation in this gene did not occur at different frequencies between asthmatics and control subjects, but was found to influence the severity of asthma (25) and it is possible that such a scenario could apply to the E237G mutation in the black population in the present study.