A comprehensive candidate gene study on bronchial asthma and juvenile idiopathic arthritis.

Bronchial asthma and juvenile idiopathic arthritis (JIA) are complex genetic diseases. As both represent chronic inflammatory diseases it is likely that they are at least partially influenced by the same genetic variants. One goal in dissecting the genetics of complex diseases is to identify a genetic risk profile. Therefore it is necessary to genotype polymorphisms in many different pathways. Thus we investigated 48 polymorphisms in 24 genes for association with asthma and/or JIA. Genotpying was performed on 231 asthmatic children, 86 children with JIA and 270 controls. Association analysis was performed by the Armitage's trend test. Furthermore haplotypes were calculated by FAMHAP. We found association of polymorphisms within IL-4, CTLA4 and TNFalpha with asthma and/or JIA. Furthermore, the polymorphisms showed an inverse distribution between children with asthma and JIA. However, we were not able to confirm association of most of the previously described candidate genes. We conclude from our data that it might be very difficult to identify genetic risk profiles for the development of asthma and/or JIA that would be valid across different populations. However, this study adds further evidence that the common genetic background of asthma and JIA is mainly based on polymorphisms in important TH1 and TH2 cytokines.

Genes for atopy and asthma.

The genetic basis of atopic diseases is represented by a complex network of interacting genes or common genetic variants rather than a few disease-causing mutations. The individual risk of developing asthma or other atopic diseases is defined by the concert of interaction of these hereditary factors and environmental stimuli. The first decade of asthma genetics has been spent identifying those genetic regions through linkage analysis, which are likely to harbour asthma genes. At the same time, several candidate genes for asthma and atopy have been identified and their variants characterized, some of them even to a level of functional understanding. Rather than adding new candidate regions and genes to the pool of knowledge, the interest in the past year has moved to a more sophisticated statistical evaluation of the given linkage and association data and a more precise definition of so-called 'intermediate phenotypes'. Some of the results are quite surprising and have helped us to understand the underlying pathophysiology. For example, the distinct clinical traits of asthma, such as atopic sensitization or inflammation of the bronchial epithelium, seem to be defined by distinct subsets of predisposing genes. At the same time, the very same subsets of genes might underlie further clinical diseases with similar clinical features. Polymorphisms within IL-4R alpha, which had been shown to be associated with asthma and atopy, have also been shown to be associated with kidney allograft rejection, systemic lupus erythematosus and Crohn's disease. There might thus just be a few asthma and atopy genes. Finally, asthma and atopy genetics has now reached a point of practical application. The genetics of susceptibility to environmental stimuli, pharmacogenetic data, and the advent of new pharmaceutical targets will greatly influence the whole field of asthma and atopy.

Recent advances in understanding how interleukin 13 signals are involved in the pathogenesis of bronchial asthma.

The prevalence of allergic disease has dramatically increased in recent decades, especially in urban and industrialized areas. Allergic diseases are disorders of the immune system, the results of complex interactions among various genetic and environmental factors. Among them, the important role of interleukin 13 (IL-13), a Th2-type cytokine, has recently emerged in the pathogenesis of bronchial asthma. Based on studies using mice, great attention has been paid to the direct effects of IL-13 on bronchial tissues. In this review, we describe recent advances in understanding the signal transduction mechanism of IL-13, the involvement of IL-13 signal-related genes as genetic factors in the pathogenesis of bronchial asthma, and the expression of IL-13 receptor on bronchial tissues. We describe potential strategies for targeting IL-13 signals to improve allergic states.

Confirmation of association of IL-15 with pediatric asthma and comparison of different controls.

BACKGROUND: Interleukin (IL)-15 is an important mediator in chronic inflammatory diseases. Recently, we have described the association of IL-15 haplotypes with bronchial asthma. Asthma genetics is highly complex - about every second candidate gene is not confirmed in consecutive studies. We were interested in whether association of asthma with IL-15 holds in a second population. Furthermore, we sought to investigate the effect of different controls. METHODS: Five IL-15 polymorphisms were genotyped on the German Multicenter Allergy Study (MAS) cohort consisting of 886 children who were followed up from birth to 10 years of age. At 10 years of age, 96 were found to be asthmatic. MAS children who never had any wheezing symptoms (n = 576), who were never diagnosed with asthma (n = 790) and 129 super controls who had never had any atopic disorder were used as controls. Finally, 270 randomly chosen adults served as controls. RESULTS: Association was confirmed with single polymorphism and haplotypes. The super controls showed the highest difference to the asthmatics regarding haplotype frequencies. However, the effect escaped statistical significance, most likely because of the small sample size. CONCLUSION: Association of IL-15 with asthma was confirmed. Although super controls might be the most suitable, more numbers are needed. This might hamper the value of these controls especially when investigating common diseases.

Association study of polymorphisms within matrix metalloproteinase 9 with bronchial asthma.

Matrix metalloproteinase 9 plays an important role in the development of bronchial asthma. We were interested in whether the polymorphisms -T1702A, -C1562T, R279Q and +C6T within the matrix metalloproteinase 9 (MMP-9) gene were associated with asthma in a population of 231 asthmatic children. However, we found no association. Thus MMP-9 might not be a major gene for asthma.

A genome-wide screen on the genetics of atopy in a multiethnic European population reveals a major atopy locus on chromosome 3q21.3.

BACKGROUND: Dissecting complex diseases in underlying distinct traits and studying these for their genetic basis might enhance the power as well as the specificity, of detection of disease genes. These phenotypes are known as intermediate phenotypes. OBJECTIVE: We were interested in the atopic basis of asthma, and used the sensitization to mite (Dermatophagoides pteronyssinus) allergens as a pathophysiologically important intermediate phenotype. METHODS: This time we performed a genome-wide scan based on the same already used multiethnic European population consisting of 82 nuclear families with at least two affected siblings. We carried out nonparametric as well as parametric MOD-score analyses based on the genotypes of 603 microsatellite markers. RESULTS: In comparison with our first genome-wide candidate region search three novel regions additionally appeared to be significant. We obtained significant results for the region 2p12 with a MOD score of 3.35 and for the region 16q21 with a MOD score of 4.18. The most significant result was found for the region 3q21.3 with the same microsatellite marker, which showed significant linkage to atopic dermatitis (AD) in another study with a MOD score of 4.51 and an nonparametric linkage analysis (NPL) of 4.00. CONCLUSION: Our findings indicate that atopy, allergic asthma, allergic rhinitis and AD on the one hand are distinct traits on both the clinical and genetic basis, but on the other hand, our results also underline that these traits are closely related diseases concerning the atopic basis of the traits.

Two common polymorphisms in the coding part of the CD43 gene are not associated with atopy.

BACKGROUND: Recently linkage and association of the chromosomal region 16p12-11 with enhanced IgE responsiveness have been shown. The gene coding for CD43 (sialophorin) has been localized to this region. Sialophorin represents a major sialoglycoprotein on the surface of human lymphocytes, monocytes and granulocytes. It is supposed to play an important role in human mast cell, T- and B-cell regulation and activation and has been described in connection with immunodeficiency diseases such as the Wiskott-Aldrich syndrome. Therefore, it can be designated as a candidate gene for atopy. METHODS: Using SSCP analysis and direct genomic sequencing, polymorphisms in the CD43 gene have been looked for and their association with atopy has been tested in a population of 260 largely atopic children and young adults. RESULTS: Three common polymorphisms in the coding part of the CD43 gene were found. Two of them are leading to amino acid exchanges, one from argine to cysteine at amino acid position 337 of the mature gene product and one from leucine to phenylalanine at amino acid position 341. Subsequent association studies revealed no obvious influence of R337C or L341F on IgE regulation (p = 0.47 and 0.43), neither in a cognate nor in an uncognate fashion. CONCLUSION: We conclude that CD43 polymorphisms are unlikely to account for the observed linkage effect at 16p12-11. Whether the polymorphisms R337C and L341F adjacent to phosphorylation sites in the intracellular region of the protein alter the normal functioning of CD43 remains to be elucidated.

Characterization of the membrane-bound and a soluble form of human IL-4 receptor alpha produced by alternative splicing.

IL-4 plays a major role in IgE production. Its signal is conferred to effector cells through binding to the alpha chain of the membrane-bound human IL-4 receptor (huIL-4Ralpha). Here we present the genomic structure and organization of huIL-4Ralpha. The promotor region shows binding sites for several transcription factors involved in inflammatory processes. HuIL-4Ralpha has been shown to be organized differently to that of mouse IL-4Ralpha. A soluble form of huIL-4Ralpha is produced by alternative splicing of the huIL-4Ralpha gene (shuIL-4Ralpha/splice). Expression of the corresponding mRNA coding for the extracellular part of the receptor and an additional three amino acids is also shown. A second form of huIL-4Ralpha, i.e. shuIL-4Ralpha/prot, is produced by limited proteolysis of the receptor (shedding) and is already known. These results reveal a complex pattern for the regulation of the IL-4 pathway at the receptor level. The patterns of expression of all three receptor proteins as well as their individual meaning in the context of inflammation still have to be elucidated.

Single region linkage analyses of asthma: description of data sets.

Linkage (genotypic) data from the 5q31-33 candidate region for asthma were contributed to Genetic Analysis Workshop 12 by members of the International Consortium on Asthma Genetics (COAG). Data came from five independent studies sampled from five countries. Genotypic data for a total of 26 markers were available, although the number of markers typed in each data set varied. Phenotypic and genotypic data was available from a total of 569 families and 3,175 subjects. The phenotypic data available varied among the studies; however information regarding physician-diagnosed asthma and total serum IgE levels was available in all five studies. This paper describes the ascertainment, data collection methods, phenotypic data, and genotypic data available for the single linkage region analyses undertaken for Genetic Analysis Workshop 12.

Common polymorphisms in the CTLA-4 and CD28 genes at 2q33 are not associated with asthma or atopy.

Recently, genetic linkage of the chromosomal region 2q33 with asthma has been shown. The genes coding for CD28 and CTLA-4 have been localized to this chromosomal region. CD28 and CTLA-4 have been shown to be involved as an important costimulatory signal in the regulation of allergic inflammation and TH2 cytokine production, and thus both genes are good candidate genes for asthma and atopy. Two common polymorphisms in the CTLA-4 gene and one polymorphism in the CD28 gene found by single-strand conformation polymorphisms (SSCP) analysis and direct genomic sequencing were tested for association with asthma and atopy phenotypes in a population of 260 largely atopic children and young adults. No association was found between any of the three polymorphisms and asthma or atopy phenotypes. The newly described common CD28 polymorphism is situated in the third intron of the gene. We conclude that neither gene is likely to exert a major influence on the development of asthma or atopy in our population. However, it might prove useful to test for association of these polymorphisms with asthma in populations recruited through asthmatic but not necessarily atopic individuals.

Common polymorphisms and alternative splicing in the ILT3 gene are not associated with atopy.

Recently, a linkage of the chromosomal region 19q13.4 with bronchial asthma has been demonstrated. This region harbours the so-called leucocyte receptor cluster with the gene for immunoglobulin-like-transcript 3 (ILT3) as a member. ILT3 represents an inhibitory receptor bearing three immunoreceptor tyrosine inhibitory motifs (ITIM). The protein mediates downregulation of cell activation through recruitment of different SH2-containing protein tyrosine phosphatases. With regard to the negative immunoregulatory function particularly on B-cells, ILT3 represents a candidate gene for atopy and asthma. The aim of this study was to screen for common polymorphisms in the gene coding for ILT3 and to test for association with the atopic phenotype. Using single-stranded conformal polymorphism-analysis and direct genomic sequencing seven polymorphisms, three mutations, a common deletion of 7 bp in the third intron and evidence for further alternative splicing of the ILT3 gene were found. Although no association was found with atopy phenotypes, it might prove useful to test for association with bronchial asthma.

Promoter polymorphisms of the CD14 gene are not associated with bronchial asthma in Caucasian children.

Several studies have investigated the association of a promoter polymorphism in CD14 with atopic phenotypes. We screened this and another polymorphism in 182 asthmatic children and found no association with asthma. Furthermore, there was substantial linkage disequilibrium of the polymorphisms. Thus CD14 does not play a major role in the development of asthma in our population of Caucasian children.

Association studies on beta2-adrenoceptor polymorphisms and enhanced IgE responsiveness in an atopic population.

The beta2 adrenergic receptor 2 represents a cell surface receptor responsible for the binding of endogenous catecholamines and their exogenously administered agonists and antagonists, mediating their effects to the interior of the cell. On the basis of these functions, the observed association of two of its polymorphisms, Gly16 and Gln27, with nocturnal- and steroid-dependent asthma has been discussed. It has recently been suggested that Gln27 contributes to IgE variability in families with asthma. The aim of this study was to investigate possible influences of the polymorphisms Arg16Gly and Glu27Gln on IgE levels in families recruited through an atopic index case without regard to the presence of clinical symptoms. We employed linkage analysis in affected sibpairs characterized by elevated total IgE concentrations or sensitization to common inhalant allergens. Furthermore, we tested 258 children for association of any of the polymorphisms with enhanced IgE responsiveness. We could find neither linkage at the locus 5q31 nor significant association of the polymorphisms with elevated total IgE concentrations or specific sensitization. We conclude from our data that the polymorphisms Gln27Glu and Arg16Gly of the beta2-adrenergic receptor do not play a significant role in the pathogenesis of enhanced IgE responsiveness in an atopic population in general.

The recognition pattern of sequential B cell epitopes of beta-lactoglobulin does not vary with the clinical manifestations of cow's milk allergy.

BACKGROUND: beta-Lactoglobulin (BLG) represents one of the major allergens causing cow's milk allergy (CMA) - a disease with a wide spectrum of clinical symptoms. The aim of this study was to evaluate sequential B cell epitopes of BLG by the Pin-ELISA method. Furthermore, we wanted to investigate a possible association of the IgE recognition patterns in sera of patients with BLG sensitization and the type of clinical reactions following contact with cow's milk. METHODS: Overlapping sequential decapeptides corresponding to the amino acid sequence of BLG were used in Pin-ELISAs specific for human IgE. Tested sera were from 14 individuals with CMA, 8 of them with a history of immediate systemic reactions and 6 with delayed skin reactions following contact with cow's milk. All of them showed specific IgE antibodies to BLG in the CAP-RAST. Control sera were from 5 healthy nonallergic individuals. RESULTS: All sera from BLG-sensitized individuals showed IgE binding with one region of BLG corresponding to amino acids 95-113. Furthermore, individual sera showed reactions with two further regions, 12-27 and 124-135. Inhibition of IgE binding to BLG with one soluble synthetic peptide confirmed the major epitope. No differences were found in the B cell epitope recognition pattern to BLG in the two groups of patients with CMA, characterized by acute systemic or delayed skin reactions. CONCLUSIONS: Using IgE Pin-ELISAs we were able to confirm previously described sequential B cell epitopes of BLG. However, the recognition pattern of one of the major cow's milk allergens is not predictive of the clinical type of reaction.

A novel polymorphism in the 5' promoter region of the human interleukin-4 receptor alpha-chain gene is associated with decreased soluble interleukin-4 receptor protein levels.

Interleukin (IL)-4 exerts its biological effects through binding to the IL-4 receptor (IL4R) complex, plays a central role in stimulating B-cell differentiation, and is crucial for the development of T helper 2 cells. Recently, a soluble form of the human IL4R alpha chain (sIL4R alpha), which is produced by alternate mRNA splicing of exon 8, was discovered. sIL4R is thought to play an important role in either enhancing or inhibiting IL-4 signalling. We analyzed the 5' promoter region of the human IL4R alpha-chain gene (IL4RA) of healthy volunteers by DNA sequencing and found three novel single-nucleotide polymorphisms (SNPs; T-890C, T-1914C, C-3223T) and one novel short tandem repeat [(CAAAA)(5-7)-3600]. The two common promoter region SNPs T-1914C and C-3223T as well as six known coding SNPs in the IL4RA gene were genotyped in healthy blood donors by PCR with sequence-specific primers; total sIL4R levels were measured by ELISA. Results revealed a highly significant association of the -3223T variant with lowered sIL4R levels (two-tailed t-test, P=0.0002). Results remained highly significant after Bonferroni adjustment for multiple comparisons (P=0.0017). Moreover, the C-3223T variant was found to be in strong linkage disequilibrium with the extracellular 150V variant (P<0.001), which was recently described to be associated with atopic asthma in a Japanese population. Since this novel IL4RA promoter region SNP is common (allele frequency 29.8%), we conclude that it may be of importance for the genetic regulation of the IL-4 signalling pathway.

Association study of polymorphisms within interleukin-18 in juvenile idiopathic arthritis and bronchial asthma.

BACKGROUND: Interleukin-18 (IL-18) plays an important role in the regulation of TH1 as well as TH2 immunologic responses and thus in the development of chronic inflammatory diseases. Positive association studies of polymorphisms in IL-18 with different diseases have underlined the involvement of IL-18 in the pathogenetics processes. Our interest was to test polymorphisms of IL-18 for association with a typical TH1-mediated disease--juvenile idiopathic arthritis--and the TH2-mediated disease bronchial asthma in Caucasian children. METHODS: We genotyped five polymorphisms that were in association with chronic inflammatory diseases (-607C, -137C, 113G, 127T, and -133G). This was performed by restriction fragment length polymorphism in populations of asthmatic children, control individuals, and children with antinuclear antibodies (ANA)-positive juvenile idiopathic arthritis. Statistical analysis was performed by the Armitage trend test; haplotypes were calculated by the Arlequine program. RESULTS: No significant association was found between any single nucleotide polymorphism or any haplotype and bronchial asthma or ANA-positive juvenile idiopathic arthritis. CONCLUSION: We conclude that the effect of IL-18 in the immunologic context of diseases like bronchial asthma or juvenile arthritis might be too complex to be reflected in a simple one-way association study. Furthermore, the polymorphisms under investigation might be nonfunctional.

Parametric and nonparametric multipoint linkage analysis with imprinting and two-locus-trait models: application to mite sensitization.

We present two extensions to linkage analysis for genetically complex traits. The first extension allows investigators to perform parametric (LOD-score) analysis of traits caused by imprinted genes-that is, of traits showing a parent-of-origin effect. By specification of two heterozygote penetrance parameters, paternal and maternal origin of the mutation can be treated differently in terms of probability of expression of the trait. Therefore, a single-disease-locus-imprinting model includes four penetrances instead of only three. In the second extension, parametric and nonparametric linkage analysis with two trait loci is formulated for a multimarker setting, optionally taking imprinting into account. We have implemented both methods into the program GENEHUNTER. The new tools, GENEHUNTER-IMPRINTING and GENEHUNTER-TWOLOCUS, were applied to human family data for sensitization to mite allergens. The data set comprises pedigrees from England, Germany, Italy, and Portugal. With single-disease-locus-imprinting MOD-score analysis, we find several regions that show at least suggestive evidence for linkage. Most prominently, a maximum LOD score of 4.76 is obtained near D8S511, for the English population, when a model that implies complete maternal imprinting is used. Parametric two-trait-locus analysis yields a maximum LOD score of 6.09 for the German population, occurring exactly at D4S430 and D18S452. The heterogeneity model specified for analysis alludes to complete maternal imprinting at both disease loci. Altogether, our results suggest that the two novel formulations of linkage analysis provide valuable tools for genetic mapping of multifactorial traits.

Linkage and association studies of atopy and the chromosome 11q13 region.

The clinical syndrome atopy is largely determined by genetic factors. In 1989, the first linkage of markers within and flanking the chromosomal region 11q13 and atopy was reported. In the following years, the gene coding for the beta chain of the high affinity IgE receptor was localised to this region and two polymorphisms in this gene have been shown to be associated with the atopic phenotype. We investigated two independent populations (population based and outpatient department) with different degrees of clinical symptoms. Using highly polymorphic markers we could find no evidence for linkage or allelic association of this particular genomic region to the atopic phenotype defined by enhanced IgE responsiveness (p>0.05). Neither did we succeed in finding either of the two polymorphisms described, nor could we identify any other polymorphisms within the gene. However, we found weak evidence for linkage in asthmatic sib pairs regarding maternal alleles (p=0.03). We conclude from our data that in our populations the gene for the beta chain of the high affinity IgE receptor is of minor importance for enhanced IgE responsiveness, and that it might influence atopy with clinical signs like asthma through maternally derived alleles.

The polymorphisms S503P and Q576R in the interleukin-4 receptor alpha gene are associated with atopy and influence the signal transduction.

Interleukin-4 (IL-4) plays a major role in immunoglobulin E (IgE) production. Its signal is conferred to effector cells through binding to the alpha chain of the IL-4 receptor (IL-4Ralpha). We present further evidence for polymorphisms in the IL-4Ralpha gene having an effect on IgE regulation. For two of four common polymorphisms, S503P and Q576R, we found an association with lowered total IgE concentrations (P=0.0008 if occurring together). The polymorphism S503P has not yet been described and is located within the I4R motif of the receptor. In vitro analyses using synthetic peptides of this region showed that the tyrosine kinase Janus kinase 1 (JAK1), as well as IRS-1 and IRS-2 bind to the I4R motif irrespective of the polymorphism or a tyrosine phosphorylation. In vivo immunoassays using T cells of four different groups of individuals (S503/Q576; P503/Q576; S503/R576; P503/R576) revealed that only in case of both polymorphisms the phosphorylation of IRS-1 and IRS-2, but not JAK1 was increased. We found no binding of STAT6 to the I4R synthetic peptides; however, the phosphorylation was reduced in the presence of any of the two polymorphisms, including P503 alone. We discuss possible conformational changes of the receptor leading to the observed effects on the phosphorylation status of IRS-1, IRS-2 and STAT6, in addition to previous findings that Q576R alters STAT6 binding. We conclude that P503 and R576 influence the signal transduction pathways through the IL-4Ralpha, an effect that is magnified by the presence of both polymorphisms. This could explain the observed association effects with lowered total IgE concentrations.