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.

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.

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.

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.

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.

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.

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.

Meta-analysis for linkage to asthma and atopy in the chromosome 5q31-33 candidate region.

Asthma is a common, complex human disease. Gene discovery in asthma has been complicated by substantial etiological heterogeneity, the possibility of genes of small effect and the concomitant requirement for large sample sizes. Linkage to asthma phenotypes has been investigated most intensively in the 5q chromosomal region, although results have been inconsistent across studies and all studies have had modest sample sizes. One potential solution to these issues is to combine data from multiple studies in a retrospective meta-analysis by pooling either summary statistics or raw data. The International Consortium on Asthma Genetics combined data from 11 data sets (n = 6277 subjects) to investigate evidence for linkage of 35 markers spanning the cytokine cluster on chromosome 5q31­33 to 'asthma' dichotomy and total serum immunoglobulin E (IgE) levels. Chromosome 5q markers typed in different centers were integrated into a consensus map to facilitate effective data pooling. Multipoint linkage analyses using a new Haseman­Elston method were performed with all data sets pooled together, and also separately with the resulting linkage statistics pooled by meta-analytic methods. Our results did not provide any evidence significant at the 5% level that loci conferring susceptibility to asthma or atopy are present in the 5q31­33 region; however, there was some weak evidence (empirical P = 0.077) of linkage to asthma affection. This study suggests that loci in 5q31­33 have at most a modest effect on susceptibility to asthma or total serum IgE levels, may not be detectable or present in all human populations and are difficult to detect even using combined linkage evidence from 2400­2600 full sibling pairs.

Studies on linkage and association of atopy with the chromosomal region 12q13-24.

BACKGROUND: Several studies have shown linkage of bronchial asthma, allergic rhinitis and total serum IgE concentration to the chromosomal region 12q13-24 in ethnical diverse populations. This region harbours a number of candidate genes for asthma and atopy, including stem cell factor (SCF), leukotriene A4 hydrolase (LTA4H), thyroid receptor 2 (TR2), and signal transducer and activator of transcription 6 (STAT6). However, the same region was shown as well to be linked to other diseases with inflammatory character. So far no variants in any of these genes have been published which would allow association studies and confirm the pathogenicity of any of these genes. OBJECTIVE: We wanted to test for linkage of the chromosomal region 12q13-24 with the atopic phenotype without regard to clinical manifestations. Furthermore we screened for common nucleotide polymorphisms in candidate genes to enable association studies. METHODS: We employed sib-pair linkage analysis and transmission disequilibrium testing with regard to four highly polymorphic microsatellite markers in 12q13-24 in atopic nuclear families. In addition, we looked for polymorphisms in the genes coding for SCF, LTA4H, TR2 and STAT6 performing SSCP-analysis and direct genomic sequencing. RESULTS: We found no evidence for linkage of the genomic region 12q13-24 to elevated total serum IgE levels, specific sensitization to common inhalant allergens or atopy. Furthermore we identified three nucleotide polymorphisms including one common variant in the gene coding for SCF. No association of this polymorphism and any of the atopic phenotypes was seen. CONCLUSION: We conclude from our data that genes in the chromosomal region 12q13-24 and in particular SCF are unlikely to exert a major effect on the induction of the atopic phenotype in our Caucasian population. However, we did not focus on the asthmatic and thereby inflammatory aspect of atopy which might explain these results in contradiction to previous studies.

A European study on the genetics of mite sensitization.

BACKGROUND: Sensitization to mite allergens represents a prominent feature of atopy and an important predictor of bronchial asthma. OBJECTIVE: It was the intention of this study to define genetic loci linked to mite sensitization because these could represent the genetic basis of the important atopic component of asthma. METHODS: We studied a multiethnic white population of 99 families, including 224 sib pairs sensitized to Dermatophagoides pteronyssinus. A genome-wide candidate-region search was performed that covered potential asthma and atopy regions. RESULTS: As for nonparametric linkage (NPL) analysis, 14 of the candidate regions showed evidence for linkage (NPL > 2.0), and 4 of them showed prominent linkage (NPL > 3.0). However, there were substantial ethnic differences. Maximizing the LOD score analysis identified candidate regions with suspected dominant and recessive mode of inheritance. Furthermore, genetic imprinting models provided significant evidence for linkage in the 8p23 region and revealed potential maternal imprinting. The regions found are distinct to those in asthma searches that have been found to be linked to asthma, as well to other inflammatory diseases. In addition, we could not find linkage to the HLA region. By different cutoff points of the phenotype definition, the IL cluster showed evidence of being linked to the degree of sensitization rather than to sensitization per se. CONCLUSION: The results indicate that the genetic basis of the atopic component of asthma is different from that of the inflammatory component. Furthermore, it seems reasonable to assume that specific sensitizations are influenced by distinct genetic variants leading to their initiation versus those leading to their enhancement.

A first trial of retrospective collaboration for positional cloning in complex inheritance: assay of the cytokine region on chromosome 5 by the consortium on asthma genetics (COAG).

The central problem of complex inheritance is to map oligogenes for disease susceptibility, integrating linkage and association over samples that differ in several ways. Combination of evidence over multiple samples with 1,037 families supports loci contributing to asthma susceptibility in the cytokine region on 5q [maximum logarithm of odds (lod) = 2.61 near IL-4], but no evidence for atopy. The principal problems with retrospective collaboration on linkage appear to have been solved, providing far more information than a single study. A multipoint lod table evaluated at commonly agreed reference loci is required for both collaboration and metaanalysis, but variations in ascertainment, pedigree structure, phenotype definition, and marker selection are tolerated. These methods are invariant with statistical methods that increase the power of lods and are applicable to all diseases, motivating collaboration rather than competition. In contrast to linkage, positional cloning by allelic association has yet to be extended to multiple samples, a prerequisite for efficient combination with linkage and the greatest current challenge to genetic epidemiology.

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.

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.

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.

Genetic variants of IL-13 signalling and human asthma and atopy.

Asthma and atopy show epidemiological association and are biologically linked by T-helper type 2 (T(h)2) cytokine-driven inflammatory mechanisms. IL-4 operates through the IL-4 receptor (IL-4R, a heterodimer of IL-4Ralpha and either gammac or IL-13Ralpha1) and IL-13 operates through IL-13R (a heterodimer of IL-4Ralpha and IL-13Ralpha1) to promote IgE synthesis and IgE-based mucosal inflammation which typify atopy. Recent animal model data suggest that IL-13 is a central cytokine in promoting asthma, through the stimulation of bronchial epithelial mucus secretion and smooth muscle hyper-reactivity. We investigated the role of common genetic variants of IL-13 and IL-13Ralpha1 in human asthma, considering IgE levels. A novel variant of human IL-13, Gln110Arg, on chromosome 5q31, associated with asthma rather than IgE levels in case-control populations from Britain and Japan [peak odds ratio (OR) = 2.31, 95% CI 1.33-4.00]; the variant also predicted asthma and higher serum IL-13 levels in a general, Japanese paediatric population. Immunohistochemistry demonstrated that both subunits of IL-13R are prominently expressed in bronchial epithelium and smooth muscle from asthmatic subjects. Detailed molecular modelling analyses indicate that residue 110 of IL-13, the site of the charge-modifying variants Arg and Gln, is important in the internal constitution of the ligand and crucial in ligand-receptor interaction. A non-coding variant of IL-13Ralpha1, A1398G, on chromosome Xq13, associated primarily with high IgE levels (OR = 3. 38 in males, 1.10 in females) rather than asthma. Thus, certain variants of IL-13 signalling are likely to be important promoters of human asthma; detailed functional analysis of their actions is needed.

The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma.

The platelet-activating factor (PAF) represents a phospholipid with complex biological functions, including involvement in inflammatory processes. The degrading enzyme PAF acetylhydrolase (PAFAH) represents a candidate for asthma and other atopic diseases. Two loss-of-function mutations of PAFAH are associated with severe asthma in Japanese individuals. Our aim was to look for further PAFAH variants in white populations, their possible association with atopic and asthmatic phenotypes, and their functional importance. We picked up three common variants in the PAFAH gene: Arg92His (exon 4), Ile198Thr (exon 7), and Ala379Val (exon 11). The known loss-of-function mutations were not seen. The variant allele Thr198 was found to be highly associated with total IgE concentrations in an atopic population (P=.009) and with "atopic asthma" in an asthmatic population (P=.008). The variant allele Val379 was found to be highly associated with "specific sensitization" in the atopic population (P=.002) and with "asthma" in the asthmatic population (P=.003). By use of recombinant PAFAH enzymes, the variant Val379 showed increased (14 microM) and Thr198 markedly increased (42 microM) KM values compared to the wild type (7 microM); furthermore, Vmax of Val379 was highly increased (132%). Thr198 and Val379 influence plasmatic PAFAH toward lower substrate affinities and therefore are very likely to prolong the activities of PAF. At the same time, they are associated with an increased risk to develop asthma and atopy. Thus, two PAFAH variants seem to play a key role in atopic and asthmatic processes in Caucasian populations.