Association of HLA-DQ2 and asthma in Brazilian children
Maria Gerbase-DeLima, Clarisse A. Gallo, Silvia Daher, Dirceu Solé, Charles K. Naspitz
Division of Allergy, Clinical Immunology and Rheumatology, Department of Pediatrics, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil
Running title: HLA and asthma
Correspondence:
Maria Gerbase-DeLima ,MD
Disciplina de Alergia, Imunologia Clínica e Reumatologia
Escola Paulista de Medicina, UNIFESP
Rua dos Otonis, 725
04025-002 São Paulo, SP, BRAZIL
Phone number: 55-11-5740548
FAX number: 55-11-5701590
e-mail: ger.dped@epm.br
ABSTRACT; INTRODUCTION; MATERIAL AND METHODS; RESULTS; DISCUSSION; REFERENCES
The purpose of the present study was to analyze the frequency of HLA-DR and DQ antigens in Brazilian asthmatic children with skin-test and RAST positivity to Dermatophagoides pteronyssimus. The comparison of HLA-DR and DQ antigenic frequencies between patients (n=30) and controls disclosed a significantly higher HLA-DQ2 frequency in the patients (60% versus 34%, P = 0.013; R.R.= 2.8).
Key words: · asthma · genetics · HLA · MHC · histocompatibility antigens · allergy·
The reason why environmental allergens induce preferential Ig E antibody responses only in a minority of individuals represents a complex question, and a further problem is to understand why only some of these individuals develop asthma. Epidemiological studies have clearly demonstrated the familial aggregation of these traits but the unraveling of the involved genetic factors has proved to be a very difficult task, since multiple genes seem to be play a role, acting at different levels. At the moment, there is evidence of genetic influence at the level of the recognition of the allergen by helper T, at the mechanisms that lead to over-production of Ig E and also at the bronchial level, favoring bronchial hyper-reactivity (1).
An interesting area of investigation is the study of polymorphisms in the HLA (the human major histocompatibility complex) region since they encode the molecules that represent the linkage elements between environmental allergens and the immune system. This interaction is initiated by the uptake, processing and presentation of allergens by class II MHC-positive accessory cells to allergen-specific helper T lymphocytes. Activated helper T cells then induce B lymphocytes to produce allergen specific antibodies mainly belonging to the Ig E class.
Some very clear associations with HLA markers have been disclosed regarding the IgE production toward some ultra-pure pollen extracts, whereas weaker associations, or no association, have been reported in studies that have analyzed the relationship between HLA and the response to more complex allergens, i.e., to mixtures of unpurified allergens or to high molecular weight allergens expressing multiple antigenic epitopes (2-4).
Considering that house dust mites (HDM) are, worldwidely, the most important allergens for inducing asthmatic symptoms in allergic adults and children (5) it is of course of considerable interest to investigate whether there is an association between particular HLA antigens and the ability to respond to HDM allergens.
A study in families with more than one asthmatic subject positive to Dermatophagoides (100% positive to D. farinae and 80% also positive for D. pteronyssinus) has shown an excess of HLA haplotypes in common among the affected subjects, suggesting the presence of an HLA-linked susceptibility gene (6).
Association studies with HLA class II antigens and HDM induced allergy have disclosed positive associations with HLA-DR3 (7) and with DQ2 (8) and a negative association with HLA-DPB1*0401 (9). Studies concerning HLA-DR and DP alleles conducted in several populations have shown positive associations of the IgE response to the purified allergen Der p I with DR4 in the Swedish and with DR3 in the Italian population, whereas no associations were found in other seven populations (3). An investigation in patients from England suggested that DR3 was positively associated with Der p II and negatively associated with Der p I IgE reactivity (4).
The purpose of the present study was to analyze the frequency of HLA-DR and DQ antigens in asthmatic children with skin-test and RAST positivity to Dermatophagoides pteronyssinus, the dominant allergen source associated with chronic asthma both in children and adults in Brazil (10).
Subjects. We studied 30 unrelated white Brazilian children (14 girls and 16 boys), aged 4 to 13 years, with severe or moderate to severe asthma. All the subjects presented positive immediate hypersensitivity cutaneous test to D. pteronyssinus extract, with Prick test reactions greater than the positive (histamine 1 mg/ml) control (wheal diameter ³ 4 mm). The levels of serum Ig E specific to D. Pteronyssinus, measured with the CAP System radioallergosorbent test (RAST) FEIA kit (Pharmacia Diagnostics, Uppsala, Sweden), were higher than 10 kUA/l in all subjects, with a mean value of 223.6, and ranging from 13 to 760 kUA/l.
HLA Typing. HLA-DR and DQ antigens were determined by the standard microlymphocytotoxicity assay and commercial anti-sera (Biotest Diagnostic, Frankfurt, Germany, Pel-Freez Clinical Systems, Brown Deer, USA) were used to define 12 HLA-DR (DR1 to DR10, DR52, DR53) and DQ1, DQ2, DQ3 specificities.
Statistical Analysis. The HLA antigenic frequencies observed in the patients were compared (Fisher’s exact test) to those determined in local ethnically matched controls. The controls consisted of unrelated Caucasian subjects (potential bone marrow donors or individuals from our HLA typing panel) and were not selected in order to exclude HDM allergens reactive subjects. In cases of significant differences (P < 0.05), the strength of the association was assessed using the odds ratio as an approximation to estimate the relative risk (R.R.).
The phenotypic frequencies of HLA-DR and DQ antigens in the asthmatics and in the control subjects are shown in Table 1. The DQ2 frequency was higher in the patients than in the control subjects (60% versus 34%, P = 0.013; R.R. 2.8). The other differences did not reach statistical significance.
Our results have shown an increased HLA-DQ2 frequency in asthmatic children with skin-test and RAST positivity to D. pteronyssinus, being this antigen present in 60% of the patients and in 34% of the controls, represented by individuals of the general population and not selected for being negative to D. pteronyssinus. Although the P value was significant (P = 0.013), one has to be aware that association studies are prone to a statistical pitfall represented by the multiple antigenic frequency comparisons that are made between patients and controls. Therefore, it is wise to multiply the P value by the number of comparisons, i.e., the number of antigens tested. After applying this correction to the P value of our study, it remained no longer significant. This does not mean that the association does not exists, but merely show that the data should not be taken as definitive. It should be noted that DQ markers were not investigated in most of the studies concerning HLA and HDM allergy. We are inclined to accept the association of DQ2 as meaningful taking into consideration that this same association was reported in asthmatic Chinese children, also selected by being reactive to D. pteronyssinus (8).
Acknowledgments
The study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado de São Paulo.