Association between the MHC gene region and variation of serum IgE levels against specific mould allergens in the horse

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Genet. Sel. Evol. 35 (Suppl. 1) (2003) S177 S190 INRA, EDP Sciences, 2003 DOI: /gse: S177 Original article Association between the MHC gene region and variation of serum IgE levels against
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Genet. Sel. Evol. 35 (Suppl. 1) (2003) S177 S190 INRA, EDP Sciences, 2003 DOI: /gse: S177 Original article Association between the MHC gene region and variation of serum IgE levels against specific mould allergens in the horse Ino CURIK a, Darrilyn FRASER b, Claudia EDER c, Roland ACHMANN d, June SWINBURNE e, Matthew BINNS e, Reto CRAMERI f, Gottfried BREM d, Johann SÖLKNER g, Eliane MARTI h a Animal Science Department, Faculty of Agriculture, University of Zagreb, Svetosimunska 25, Zagreb, Croatia b Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington , USA c Division of Immunogenetics, Institute of Animal Breeding, University of Berne, Bremgartenstrasse 109 A, 3012-Berne, Switzerland d Ludwig-Boltzmann-Institute for Cyto-, Immuno- and Molecular Genetic Research, Veterinärplatz 1, 1210-Vienna, Austria e Animal Health Trust, Newmarket, Suffolk CB8 7UU, UK f Swiss Institute of Allergy and Asthma Research (SIAF), Obere Strasse 22, 7270-Davos, Switzerland g Department of Livestock Science, University of Agricultural Sciences, Gregor-Mendel-Strasse 33, 1180-Vienna, Austria h Division of Clinical Immunology, Department of Clinical Veterinary Medicine, University of Berne, Länggass-Strasse 124, 3012-Berne, Switzerland (Accepted 26 February 2003) Abstract To investigate whether the equine major histocompatibility complex (MHC) gene region influences the production of mould-specific immunoglobulin E antibodies (IgE), alleles of the equine leukocyte antigen (ELA-A) locus and three microsatellite markers (UM-011, HTG-05 and HMS-42) located on the same chromosome as the equine MHC were determined in 448 Lipizzan horses. Statistical analyses based on composite models, showed significant associations of the ELA-A and UM-011 loci with IgE titres against the recombinant Aspergillus fumigatus 7 antigen (rasp f 7). UM-011 was also significantly associated with IgE titres against the recombinant Aspergillus fumigatus 8 antigen (rasp f 8). In addition to the loci mentioned above, the MHC class II DQA and DRA loci were determined in 76 Lipizzans from one stud. For IgE levels against rasp f 7, the composite model showed the strongest association for DQA Correspondence and reprints S178 I. Curik et al. (P 0.01) while for rasp f 8 specific IgE levels, similarly to the results found with all 448 horses, the strongest association was found with UM-011 (P = 0.01), which is closely linked with the MHC class II DRB locus. These results suggest that the equine MHC gene region and possibly MHC class II loci, influence the specific IgE response in the horse. However, although the strongest associations were found with DQA and UM-011, this study did not distinguish if the observed effects were due to the MHC itself or to other tightly linked genes. horse / major histocompatibility complex / immunoglobulin E / specific mould allergen 1. INTRODUCTION Immunoglobulin E (IgE) is an antibody class which mediates immediate type hypersensitivity reactions and plays an important role in the pathogenesis of allergic diseases and in the defence against helminths. Allergic diseases are hypersensitivity reactions of certain individuals to substances called allergens, often present in the natural environment, which are usually harmless for the majority of the population. Production of specific IgE is initiated by the uptake and presentation of allergens by major histocompatibility complex (MHC) class II positive cells to allergen-specific helper T lymphocytes. These T lymphocytes instruct B lymphocytes to produce allergen-specific IgE antibodies. Allergen-specific IgE antibodies bind to the high affinity receptors present on mast cells and basophils and allergen-induced IgE cross-linking results in the release of various inflammatory mediators. In human studies of family aggregation and segregation, it has been suggested that total serum IgE levels are under genetic control and are associated with, or linked to a number of genes [2], although the mechanism of inheritance has not been determined. In addition to genetic factors which influence a general predisposition for high IgE levels, MHC class II genes, and more precisely HLA-D genes, are involved in the specific immune responsiveness to allergens [2]. Family studies have also demonstrated a genetic predisposition for IgE-mediated allergic diseases, such as asthma in humans [5]. In the horse, recurrent airway obstruction (RAO), which has some similarities with human asthma, is caused by hypersensitivity reactions to moulds and possibly also to other components present in hay and straw dusts [19]. IgE-mediated immune responses are probably involved in the pathogenesis of RAO: affected horses have higher IgE levels against mould extracts [12,21] and against certain recombinant (r) mould allergens [6] than healthy control horses. A genetic predisposition for RAO has been demonstrated with family studies [15], but an association between the equine leukocyte antigens (ELA) and RAO has not been found [15]. This lack of association is not surprising, since RAO is probably caused by immune reactions against a large number of different antigens and associations with the MHC are only found with specific IgE responses against single, well-defined allergenic molecules [2]. Equine MHC and specific IgE levels against moulds S179 A recent study in Lipizzan horses has shown that allergen-specific serum IgE levels are influenced by environmental and genetic factors [7]. A preliminary investigation also suggested that some MHC class I alleles, also called equine leukocyte antigens (ELA), are associated with the IgE response against mould allergens [7]. In order to further investigate whether the MHC gene region influences the allergen-specific IgE response in the horse, three microsatellite markers located on the same chromosome (ECA-20) as the equine MHC [23] were typed on 448 Lipizzan horses from six national studs. One of these markers, UM-011, is closely linked with the MHC class II DRB locus [23]. Furthermore, alleles of the MHC class II DQA and DRA loci were determined in 76 Lipizzan horses from one stud. IgE levels against two mould extracts and against three recombinant mould allergens had been determined previously in the sera of these horses [7]. 2. MATERIALS AND METHODS 2.1. Horses Four-hundred and forty-eight Lipizzan horses, brood mares and stallions, belonging to six studs from six different countries were included in the study: Austria: Piber stud (153 horses); Croatia: Djakovo stud (56 horses); Italy: Monterotondo stud (61 horses); Hungary: Szilvásvárad stud (76 horses); Slovakia: Topol cianky stud (42 horses); and Slovenia: Lipizza stud (59 horses). All tested horses belonged to two generations (102 parent-offspring pairs) and were over three years old. A detailed description of the population structure is reported in Zechner et al. [25]. Within each stud, the horses were exposed to similar environmental conditions and fed the same hay. All mares were kept loose in group housing stables during the winter and were at pasture and in stables during the summer. The stallions were held in individual loose boxes in stables separate from the mares, but were fed the same hay. A clinical examination of the respiratory tract permitting to identify horses suffering from RAO could unfortunately not be carried out because the brood mares of a number of studs were not used to handling, rending a thorough examination of the respiratory tract impossible without sedation. Although we did not see horses severely affected with RAO, coughing animals were present, but we could not exclude infections of the respiratory tracts and thus did not include the diagnosis as a variable in the statistical analysis. We can expect from previous studies [19] that at least 10% and probably more horses were affected with RAO Blood samples Blood was taken from the jugular vein from each horse with sodium heparin containing vacutainers for the serological MHC typing, with EDTA containing S180 I. Curik et al. vacutainers for DNA isolation and with vacutainers (Vacuette Greiner, Huber u. Co., Reinach, Switzerland) containing a serum clot activator for preparation of the serum. Serum was frozen in aliquots within 24 h after blood sampling andthenstoredat 70 C until assayed Serological MHC class I typing Alloantisera detecting 17 internationally recognised (A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, W11, A14, A15, A16, A17, A18, A19 and A20) and six locally defined (Be22, Be25, Be27, Be28, Be30 and Be108) ELA-A locus specificities were tested in a classical two-step microcytotoxicity test in Terasaki plates according to Lazary et al. [13,14] Isolation of DNA DNA was isolated from whole blood using a Nucleon DNA Extraction Kit (Amersham Biosciences UK Limited, Buckinghamshire, England) following the manufacturer s instructions Determination of MHC class II alleles MHC class II DRA and DQA locus typing was performed by the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of exon 2 according to previously published protocols [1,10] in the 76 horses from the Szilvásvárad stud. Typing for DRB [9] was performed but the data was not included in the analysis because of unreliable results Genotyping of microsatellite markers Microsatellite markers UM-011, HTG-05 and HMS-42 mapping to equine chromosome 20 (ECA-20; Swinburne et al. [23]) were genotyped in the 448 horses. While the UM-011 locus is placed within MHC class II (no recombination between UM-011 and DRB [23]), the other two microsatellites (HTG-05 and HMS-42), here used as flanking controls, are located proximal and distal on ECA-20. The three primer pairs (http://www.aht.org.uk/genetics/table2.html and were pooled into a PCRmultiplex performed as described in Swinburne et al. [23] Determination of allergen-specific IgE levels in the horse Serum IgE levels against Alternaria alternata (Alt a)and Aspergillus fumigatus (Asp f ) extracts, and against recombinant Alternaria alternata 1 (ralt a 1), recombinant Aspergillus fumigatus 7 and 8 (rasp f 7 and rasp f 8) were determined by enzyme linked immunoassay (ELISA) as described previously [6,7]. Equine MHC and specific IgE levels against moulds S Allele frequencies and associations between ECA-20 loci Allele frequencies were derived from the genotypic frequencies. Assumptions of the Hardy-Weinberg equilibrium (HWE) were tested for each locus within studs by the Markov-chain Monte Carlo algorithm implemented in the Genepop program version 3.2a [18], which generates exact tests for multiple alleles [11]. Bonferoni corrections (six comparisons per locus) were used on the HWE tests to reduce the Type I error rate. Associations between ECA-20 loci were presented as correlation coefficients, i.e. unbiased linkage disequilibrium coefficients normalised by allele frequencies, between combinations of loci or alleles included in the study and were statistically tested according to Weir [24]. The analysis was performed by the stud as well as on the pooled sample with all horses. Both the calculation of the correlation coefficients and their significance were performed by GENETIX 4.02 [3] Effects of ECA-20 loci on IgE level Variance analyses of the IgE levels were performed by the SAS statistical package [20]. General linear models (PROC GLM) were used for normally distributed variables, serum IgE levels against the mould extracts Alternaria alternata (Alt a) andaspergillus fumigatus (Asp f ). The recombinant mould allergens ralt a 1, rasp f 7 and rasp f 8 were treated as binary variables (detectable or undetectable IgE level) and were analysed with generalised linear models (PROC GENMOD) assuming a binomial error distribution [22]. In all models, response variables were analysed separately (univariate approach). The following procedure was applied in all cases. First, we fitted a model that included the effects of the stud (fixed), sex (fixed), age at measurement (covariable) and all possible two-way interactions between these effects. Then, starting from the higher order terms to lower order terms, the predictor variables having the highest P value were omitted from the model if they were nonsignificant. Later, full-composed genetic models including all loci effects were simultaneously fitted on the minimal non-genetic models. Repeating the same modelling strategy as before (reduction of non-significant effects with the highest P value), we built the resultant genetic model. A description of the models used in the analysis is shown in Table I. The effects of the ECA-20 loci on the IgE response against the mould extracts as well as against the recombinant mould allergens were tested by gene substitution models [8]. Dependences that occured in a gene substitution model were removed [16]. All rare alleles with a frequency of less than 4% were pooled (see Tabs. IV and VI). This procedure enabled us to reduce the chance of making a type I error. The same modelling strategy (for description of the models used see Tab. I) was repeated for the sample with the 76 horses from the Szilvásvárad stud with addition of two MHC class II loci (DQA and DRA). S182 I. Curik et al. Table I. Description of the models used in the analysis with respect to the non-genetic effects, ECA-20 loci involved, statistical analysis (logistic and linear regression), response variables (Alt a, Asp f, ralt a 1, rasp f 7 and rasp f 8) and dataset used (all studs and Szilvásvárad stud only). Effects Linear regression Logistic regression Alternaria a. Aspergillus f. ralt a 1 rasp f 7 rasp f 8 All studs (N = 448) Stud Age ns ns ns Sex ns ns ns ns ns Stud Age ns ns Stud Sex ns ns ns ECA-20 loci ELA-A, HMS-42, HTG-05 and UM-011 Szilvásvárad (N = 76) Age ns ns ns ECA-20 loci DQA, DRA, ELA-A, HMS-42, HTG-05 and UM-011 Significance of non-genetic effects: ns = not significant, P 0.05, P 0.01 and P N = number of horses. Since the number of horses was much lower (76), in these models, all rare alleles with a frequency of less than 5% were pooled (see Tabs. V and VII). Overdominance effects were tested according to the symmetrical overdominance model [17]. Under symmetrical overdominance model values of homozygotes were compared against heterozygotes. However, there was no single significant association observed and, thus, the results related to this model are not further presented. 3. RESULTS 3.1. Population genetics of the ECA-20 loci Fifteen different equine leukocyte antigen A (ELA-A) alleles were detected on the typed Lipizzan horses (Tab. IV). At the DQA locus, four alleles out of eight accounted for 94% of all alleles (see Tab. V). At the DRA locus, three alleles were identified; DRA*0101 had the highest allele frequency (53.3%), followed by DRA*0201 (33.8%) and DRA*0301 (11.9%). Amongst the three microsatellite loci (HTG-05, UM-011 and HMS-42), the largest number of alleles (10) was observed at the UM-011 locus. Four alleles (161, 163, 165 and 177) with frequencies ranging from 14.1% to 32.8% accounted for 89.8% of all alleles, (Tab. VI). Only five and four alleles were found at the HTG-05 and HMS-042 loci, respectively. At both loci, two alleles were predominant. The HTG and HTG alleles appeared with frequencies of 35.9% Equine MHC and specific IgE levels against moulds S183 Table II. Correlation coefficients (normalised disequilibrium coefficients) between loci on equine chromosome 20 (ECA-20). Locus HMS-42 HTG-05 UM-011 ELA-A DQA All studs (N = 448) ELA-A HMS HTG Szilvásvárad (N = 76) DRA HMS HTG UM ELA-A P 0.05, P 0.01 and P N = number of horses. and 53.1%, respectively. A similar situation was observed at the HMS-042 locus; the frequencies of the HMS and HMS alleles were 40.2% and 47.6%, respectively. All loci at all studs were in agreement with Hardy-Weinberg equilibrium. ELA-A and UM-011 as well as ELA-A and HTG-05 were found to be in significant linkage disequilibrium (Tab. II). Analysis of pairwise allele associations showed significant correlations between certain alleles. For example, between alleles ELA-A1 and UM , ELA-A8 and UM , ELA-A14 and UM , and ELA-A16 and UM significant (P ) correlation coefficients of 0.451, 0.865, and were observed, respectively. For the small dataset (76 horses), the highest significant correlation coefficients were found between the two MHC class II loci, DQA and DRA, as well as between DRA and UM-011, anddqa and UM-011. Furthermore, highly significant correlation coefficients were observed between ELA-A and DQA, ELA-A and UM-011,andELA-A and DRA (Tab. II) Association between ECA-20 loci and IgE levels against the mould extracts No significant overall effect (gene substitution model) of any of the loci considered was found for the IgE serum against the mould extracts Alt a and Asp f Association between ECA-20 loci and IgE levels against the recombinant mould allergens Although, for the model with 76 horses (Szilvásvárad stud), the effect of microsatellite UM-011 nearly reached the level of significance (P = 0.054), S184 I. Curik et al. Table III. Associations between the effects of different loci on equine chromosome 20 (ECA-20) and serum IgE (detectable or undetectable) against the recombinant mould allergens: raspergillus fumigatus 7 and 8 (rasp f 7 and rasp f 8). rasp f 7 rasp f 8 Loci df deviance P-value deviance P-value All studs (N = 448) HMS (2) (1) HTG (1) (2) UM ELA-A (3) Szilvásvárad (N = 76) HMS (3) (2) HTG (1) UM (2) ELA-A (4) (1) DQA (4) DRA (5) (3) Status of the locus (in parentheses) relates to the order of deletion of certain loci from the model: (1) = first deleted,...,(5) = fifth deleted. N = number of horses. no significant overall loci effects on serum IgE levels against ralt a 1 were obtained (data not shown). On the contrary, significant overall loci effects (Tab. III) were obtained when the analysis was performed for serum IgE levels against rasp f 7 or rasp f 8. When the analysis was performed with the 448 horses, UM-011 and ELA-A were significantly associated (P 0.05) with the presence or absence of IgE serum against rasp f 7. Significant positive effects were detected for alleles UM and ELA- A1, while a significant negative effect was detected for the ELA-A8 allele (Tab. IV). In the model including only the 76 horses from Szilvasvarad stud, a significant association (P 0.01) was obtained between the overall effect of DQA and the IgE response to rasp f 7. As shown in Table V, a significant positive effect (P 0.01) was observed for the DQA*0301 allele. A significant association was also obtained between microsatellite UM-011 and serum IgE against rasp f 8 (Tab. III) when the horses from all studs were analysed. Again, as was the case for the IgE response to rasp f 7, the UM allele was significantly positively associated with IgE against rasp f 8 (Tab. VI). In addition, a significant negative effect was observed (Tab. VI) for the UM allele as well as for the pooled rare alleles. Equine MHC and specific IgE levels against moulds S185 Table IV. Gene substitution effect (α) for ELA-A and microsatellite UM-011 loci on the IgE response to recombinant Aspergillus fumigatus [rasp f 7] in the model with 448 horses. Locus Allele # Frequency α (SE) ELA-A A (0.31) A (0.44) A (0.49) A (0.46) A (0.39) A (0.61) A (0.48) A (0.35) A (0.30) Be (0.36) Pooled rare alleles (0.42) UM (0.23) (0.28) (0.20) (0.26) Pooled rare alleles (0.37) Gene substitution effects (α) are derived from a generalised linear model assuming a binomial error distribution. P 0.05, P # For the microsatellite marker UM-011 the alleles relate to the size in bp. Pooled rare alleles: ELA-A (A10, A11, A17, A20 and Be30) and UM-011 (159, 164, 167, 171, 172 and 173). Table V. Gene substitution effect (α) for the DQA locus on the IgE response to recombinant Aspergillus fumigatus [rasp f 7] in the model with 76 horses. Allele Frequency α (SE) DQA* (0.47) DQA* (0.28) D
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