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Keywords:

  • atopy;
  • interleukin-4 receptor;
  • Ile50Val polymorphism;
  • Singapore

A topy is the genetic predisposition to the development of high levels of IgE against normally innocuous allergens, and it frequently manifests clinically as allergic asthma, eczema, or rhinitis. In a Japanese population, homozygotes for the Ile50 allele polymorphism in the extracellular domain of the α chain of the interleukin-4 receptor complex (IL-4Rα) have been found to be in excess among the atopics, leading the authors to conclude that IL-4Rα is a major atopy locus ( 1). To confirm this association, we determined the frequency of the Ile50Val genotype in relation to atopy in the three main ethnic groups in the Singapore population.

Subjects were recruited from patients attending the Asthma Clinic of the Children's Medical Centre, National University Hospital. Atopy was defined as the presence of any clinical symptom of allergic asthma, allergic rhinitis, or atopic eczema, either a total serum IgE level 2 SD above mean for age ( 2), and/or the presence of sensitization to the house-dust mite Dermatophagoides pteronyssinus as demonstrated by skin prick test or FAST. Controls were volunteers recruited from hospital and institute staff and were defined as individuals without any history of the above-mentioned atopic diseases, a normal serum IgE level corrected for age, and negative reaction to D. pteronyssinus on FAST.

Genomic DNA was extracted from blood with the QIAamp Blood Kit (QIAGEN GmbH, Germany). Separate PCR reactions were performed with the two allele-specific sense primers (corresponding to nucleotide positions 383–398) and a common antisense primer 5′-CGCTGGGCTTGAAGGAG-3′ corresponding to nucleotide positions 514–530 in the IL-4 receptor gene. The sequence for the Val50-specific primer was 5′-GAAGCCCACACGTGTG-3′; for Ile50, it was 5′-GAAGCCCACACGTGTA-3′. Amplifica-tion products were detected by ethidium bromide staining after electrophoresis through a 2% NuSieve gel. To confirm the accuracy of genotyping, we performed cycle sequencing on 13 samples. The region of interest was amplified with AmpliTaq Gold and primers 5′-CGGAATTCCGAGGCCCACACGTGT-3′ and 5′-CGCTGGGCTTGAAGGAG-3′. Bands were excised from agarose gels after electrophoresis and purified with the QIAquick Gel Extraction Kit (QIAGEN GmbH, Germany). Cycle sequencing was performed with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, USA). Products were analyzed with the ABI 377 Genetic Analyzer. Data from sequencing and AS-PCR genotyping concurred in all cases.

A total of 241 individuals (108 controls, 133 atopics) from the three major ethnic groups in Singapore were genotyped for this polymorphism. Association analyses were performed separately for each ethnic group. Fisher's exact tests were used for comparing distribution and detecting associations. The allelic distributions conformed to the Hardy–Weinberg equilibrium.

Frequency distributions of the three genotypes are presented in Table 1. We were unable to replicate Mitsuyasu et al.'s ( 1) finding of an excess of homozygotes for the Ile50 allele among the atopics. Instead, there was overrepresentation of the Val50 allele in the atopic group compared to the controls, but this was statistically significant only for the Indians (P<0.05). Ile50 was found to be the more abundant allele for controls in all three groups, whereas it was the rarer allele for Japanese controls ( 1). Interethnic differences in both allelic frequency and genotype distribution were also observed across the three ethnic groups.

Table 1.  Frequency of IL-4 receptor Ile/Val genotypes in normal controls and atopic individuals
          
  Genotype frequencyHardy– Weinberg equilibrium Genotype frequency comparison between atopics and controls  Allele frequency Atopics and controls (within ethnic groups) Atopics (between ethnic groups)
Population (n)  Ile/IleIle/ValVal/Valχ2P valueFisher's P valueIle50Val50Z-score (P value)Z-score (P value)
  1. NS: not significant at α=0.05.

ILE–VAL
All ethnic groups
 Normals (108)obs46 (42%)42 (39%)20 (19%)3.28NSP=0.156 (NS) 0.6200.3802.09 (P<0.05)  
 exp41.650.915.6
 Atopics (133)obs42 (31%)56 (42%)35 (26%)3.22NS 0.5260.474
 exp36.866.329.8
Singaporean Chinese          Chinese vs Indian
 Normals (70)obs24 (34%)30 (43%)16 (23%)1.21NSP=0.274 (NS) 0.5570.4431.65 (NS)1.96 (P<0.05)
 exp21.734.513.7
 Atopics (78)obs21 (27%)30 (38%)27 (35%)3.99NS 0.4620.538
 exp16.638.822.6
Singaporean Indian          Indian vs Malay
 Normals (17)obs11 (65%) 5 (29%) 1 (6%)0.17NSP=0.278 (NS) 0.7940.2061.85 (0.1<P<0.05) 0.06 (NS)
 exp10.7 5.6 0.7
 Atopics (26)obs11 (42%)10 (39%) 5 (19%)0.91NS 0.6150.385
 exp 9.812.3 3.8
Singaporean Malay          Malay vs Chinese
 Normals (21)obs11 (53%) 7 (33%) 3 (14%)1.02NSP=0.309 (NS) 0.6900.3100.730 (NS)2.12 (P<0.05)
 exp10.0 9.0 2.0
 Atopics (29)obs10 (35%)16 (55%) 3 (10%)0.85NS 0.6210.379
 exp11.213.7 4.2

Ethnic differences in genetic susceptibility to asthma and atopy have been suggested by genome-wide scans which have identified different susceptibility loci in various populations ( 3). Association studies with candidate genes/susceptibility loci followed similar trends ( 4, 5). Similar results were obtained with another polymorphism in a different region of the same gene, where the R576 allele was found to be associated with atopy in Caucasians, but not in Japanese ( 6, 7). Such ethnic variations may explain the disparity in our results and highlight the need to discern those susceptibility genes relevant to each population.

Acknowledgments

  1. Top of page
  2. Acknowledgments
  3. References

This work was supported by MFRC grant RP3690026 and IMCB core funding. We thank T. Shirakawa for sharing unpublished data on PCR conditions and primer sequences.

References

  1. Top of page
  2. Acknowledgments
  3. References
Footnotes
  1. There was no association in Chinese, Malay, and Indian atopics.