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No linkage or association of the IL-4Rα gene Q576R mutation with atopic asthma in Italian families
  1. * Section of Biology and Genetics, DMIBG, University of Verona, Strada Le Grazie 8, 37134 Verona, Italy
  2. Section of Paediatrics, DMIBG, University of Verona, Polyclinic “G B Rossi”, Verona, Italy
  3. Section of Paediatrics, Hospital of Bolzano, Italy
  4. § Section of Immunology, Department of Pathology, University of Verona, Italy

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    Editor—Atopy, a familial clinical syndrome of asthma, rhinitis, and eczema, is characterised by IgE mediated allergy, which results from genetic and environmental events. The disease is immunologically defined by the presence of specific IgE antibodies to common allergens and raised total serum IgE concentrations. Also, because of its high prevalence, age dependent penetrance, and assumed heterogeneity, the mode of inheritance is unknown.1 Several genes which affect IgE responsiveness have been reported.2 Among the genes that have been reported to affect the atopic phenotype, that for IL4 regulates the production of IgE.3 4 Through its receptor (IL4-R), IL4 signals target cells and tissues to mount a response. The IL4-R α chain binds IL4 and mediates its effect through kinases attached to the intracellular domain.5 TheIL4-Rα coding gene has been localised to the short arm of chromosome 16 (16p12.1).6 Sharing of maternal markers flanking the IL4-Rα gene was recently found in atopy.7 A gain of function mutation in the IL4-Rα gene was recently described, and it was reported to be associated with higher levels of expression of CD23 by IL-4, severe atopic dermatitis, raised serum IgE level, or a specific response to a common allergen in a case-control study with 20 affected and 30 unaffected adults in the North American population.8 This association study needs confirmation in a different sample.

    In the present study we have investigated the frequency of theIL4-Rα Q576R mutation in a large sample of Italian atopic asthmatic families to determine its involvement in genetic susceptibility to some atopic asthma related phenotypes.

    A panel of 851 subjects was analysed, recruited from 192 families with one or more affected children, of whom 133 were attending the Allergy and Pulmonology Clinic of the Department of Paediatrics of the University of Verona, as described previously,9 and 59 families attending the Institute of Paediatrics of the Hospital in Bolzano, both located in north east Italy. All the subjects were tested for clinical history, total serum IgE level, skin prick test (SPT), and bronchial hyper-responsiveness (BHR). Clinical asthma was defined according to the American Thoracic Society criteria,10including the response to a respiratory questionnaire. BHR to methacholine was defined as PC 20<25 mg/ml. Atopy was defined by the presence of one or both of the following criteria: (1) positive SPT to one or more common aeroallergens, such as house dust mites, cat, dog,Alternaria, grass pollen,Parietaria; (2) raised circulating total IgE (from 0 to 10 years of age: age adjusted standard curve, levels above the 90th centile; after 10 years of age: 200 kU/l). Among the families, 522 atopic subjects, 477 with a positive SPT, 288 with raised total serum IgE levels, 298 with clinical asthma, and 281 with BHR were analysed. The total is greater than the total number of people, as two or more phenotypes could occur together in the same person.

    Genomic DNA was extracted from whole blood, according to standard protocols. To achieve an easy and rapid evaluation of the guanine for adenine substitution at nucleotide 1902 of theIL4-Rα gene, a new protocol based on RG-PCR and nuclease digestion11 was developed. This method is faster than the single strand conformation polymorphism technique (SSCP) and DNA sequencing analysis previously described.8 A mutant oligonucleotide which inserted in the product of DNA amplification a new restriction site for DdeI was synthesised. The primers used were a novel 21-mer forward oligonucleotide (5′-TCGGCCCCCACCAGTGGCTCT-3′) with an A1899C substitution, and the same reverse oligonucleotide previously described.8 The PCR product is a 298 bp fragment. Restriction analysis with DdeI results in two fragments of 277 and 21 bp for the Q576 allele, and a single fragment of 298 bp for the 576R allele, as shown in fig 1. All the subjects were genotyped. The data were analysed using the affected sib pairs method implemented in SIBPAL (SAGE package) and the transmission disequilibrium test.14 The frequency of the 576R mutation calculated in the family founders was 18% (PIC=0.25). In the normal controls, the allele R frequency was 17.5% (18/104 alleles). This frequency is similar to that reported for US controls (10%).8

    Figure 1

    Detection of the IL4-Rα mutation with DdeI restriction analysis of PCR products after modification of the restriction site during PCR. (Top) Pedigree of family BZ78. Filled symbols denote affected family members. (Bottom) Electrophoretic analysis. Lanes 1-3: family members shown above, genotypes are QR, RR, QQ, respectively; lane 4: QR control. M: molecular weight marker (1 kb ladder, GIBCO-BRL).

    Linkage analysis indicated no significant increase in allele sharing for atopy, SPT, IgE, BHR, or asthma, respectively. No allele transmission disequilibrium was observed. Association studies were performed with 52 adult unrelated controls in whom positivity for each of the phenotypes was excluded. Cases were collected from family founder members according to the phenotype (asthmatic: 45, BHR: 60, IgE: 71, SPT: 153, atopy: 175). The case-control analysis did not show a variation in the distribution of alleles between cases and controls in the phenotypes investigated. The statistical tests have sufficient power (80%) to detect an association with an odds ratio of 3 for IgE, SPT, atopy, and of 5 for asthma and BHR.

    As the informativity of Q576R was limited (PIC 0.25), further linkage analyses with additional IL4-Rαpolymorphisms13 may better define the putative involvement of the gene in hereditary susceptibility to atopic asthma. Wang et al 14 have recently noted that mutation Q576R does not have a direct effect on IL-4 signal transduction in murine cells and suggested that the hypersensitive induction of CD23 in cells derived from human allergy patients may be the result of different or additional alterations in the IL-4 signalling pathway. Another IL4-Rα gene mutation, Ile50Val, was reported to be associated with atopic asthma, increased total IgE, and increased mite specific IgE in a Japanese population, and to upregulate B lymphocyte growth and IgE production in response to IL4 challenge.15 16 This mutation will now be investigated in the Italian group of patients.


    The results reported in this paper were partially obtained by using the program package SAGE, which is supported by a US Public Health Service Resource grant (1 P41 RR03655) from the Division of Research Resources. This work was partially funded by Telethon Italy, Italian Ministry of University and Research, Italian CNR Target Project Biotechnology, and Consorzio Studi Universitari Verona and Italian CNR contribution N.99.02567.CT04 to PFP.