A second-generation genomewide screen for asthma-susceptibility alleles in a founder population

A genomewide screen for asthma- and atopy-susceptibility loci was conducted, using 563 markers, in 693 Hutterites who are members of a single 15-generation pedigree, nearly doubling the sample size from the authors' earlier studies. The resulting increase in power led to the identification of 23 loci in 18 chromosomal regions showing evidence for linkage that is, in general, 10-fold more significant (P<.001 vs. P<.01) than the linkages reported previously in this population. Moreover, linkages to loci in 11 chromosomal regions were identified for the first time in the Hutterites in this report, including five regions (5p, 5q, 8p, 14q, and 16q) showing evidence both of linkage, by the likelihood ratio (LR) chi(2), and of disequilibrium, by the transmission/disequilibrium test. A region on chromosome 19 continues to show evidence for linkage, by both tests, in this study. Studies of 17 candidate genes provide evidence for association with variation in the IL4RA gene (16p12), the HLA class II genes (6p21), and the interferon-alpha gene cluster (9p22), but the lack of evidence for linkage in these regions by the LR chi(2) test suggests that these are minor susceptibility loci. A polymorphism in the CD14 gene is in linkage disequilibrium with an as yet unidentified susceptibility allele in the 5q cytokine cluster, a region showing evidence for linkage among the Hutterites. Finally, 10 of the regions showing evidence for linkage in the Hutterites have shown evidence of linkage to related phenotypes in other genome screens, suggesting that these regions may contain common alleles that have relatively large effects on asthma and atopy phenotypes in diverse populations.     

Gene-Gene Interaction in Asthma: IL4RA and IL13 in a Dutch Population with Asthma

Asthma is a common respiratory disease that is characterized by variable airways obstruction caused by acute and chronic bronchial inflammation; associated phenotypes include bronchial hyperresponsiveness (BHR), elevated total serum immunoglobulin E (IgE) levels, and skin tests positive to common allergens. Binding of interleukin-13 (IL13) or interleukin-4 (IL4) to the IL4 receptor (IL4R) induces the initial response for Th2 lymphocyte polarization. Both IL13 and IL4 are produced by Th2 cells and are capable of inducing isotype class-switching of B-cells to produce IgE after allergen exposure. These cytokines also share a common receptor component, IL4R alpha. We have investigated five IL4RA single-nucleotide polymorphisms in a population of Dutch families ascertained through a proband with asthma. By considering the probands and their spouses as an unrelated sample, we observed significant associations of atopy and asthma-related phenotypes with several IL4RA polymorphisms, including S478P and total serum IgE levels (P=.0007). A significant gene-gene interaction between S478P in IL4RA and the -1111 promoter variation in IL13, previously shown to be associated with BHR (P=.003), was detected. Individuals with the risk genotype for both genes were at almost five times greater risk for the development of asthma compared to individuals with both non-risk genotypes (P=.0004). These data suggest that variations in IL4RA contribute to elevated total serum IgE levels, and interaction between IL4RA and IL13 markedly increases an individual's susceptibility to asthma.     

Mapping and identifying genes for asthma and psoriasis

Susceptibility genes for complex diseases are characterized by reduced penetrance, caused by the influence of other genes, the environment or stochastic events. Recently, positional cloning efforts have yielded several candidate susceptibility genes in different complex disorders such as Crohn's disease and asthma. Within a genetic locus, however, the identification of the effector gene may pose further challenges and require functional studies. I review two examples of such challenges: the cloning of GPR154 (GPRA) and AAA1 on chromosome 7p14 at a susceptibility locus for atopy and asthma, and the study of HLA-Cw6, CCHCR1 (HCR) and CDSN on chromosome 6p21 at PSORS1, the major susceptibility locus for psoriasis. The susceptibility locus for atopy and asthma contains two genes and only one of them is protein coding. We studied its isoform-specific expression in bronchial biopsies and in a mouse model of ovalbumin-induced inflammation of bronchial epithelia. In the PSORS1 locus, strong linkage disequilibrium between genes has made it difficult to distinguish the effects of the three nearby genes. We engineered transgenic mice with either a HCR non-risk allele or the HCR*WWCC risk allele controlled by the cytokeratin-14 promoter. The results suggested that the overexpression of HCR in mouse skin was insufficient to induce a psoriasiform phenotype, but it appeared to induce allele-specific gene expression changes that were similar to those observed in psoriatic skin.     

<Emphasis Type="Italic">IL4</Emphasis> in the 5q31 context: association studies of type 1 diabetes and rheumatoid arthritis in the Spanish population

IL4, the gene coding the prototypic Th2 cytokine, has been frequently studied in the context of several inflammatory conditions, but conclusive results have not been obtained. This gene is located in the 5q31-33 complex genetic region, which shows some susceptibility factors to type 1 diabetes (T1D) and rheumatoid arthritis (RA) among other inflammatory conditions. Our aim was to assess the involvement on T1D and RA of IL4 polymorphisms considered individually and in combination with other polymorphisms in 5q31-33, specifically in the OCTN locus, where the L503F polymorphism has been associated with Crohn's disease and other Th1 diseases. We performed a case-control study including 316 T1D patients, 599 RA patients and 540 healthy controls, all of them corresponding to white Spanish individuals. The IL4 single-nucleotide polymorphisms (SNPs) -590C/T (rs2243250) and the OCTN1 exonic SNP L503F (rs1050152) were analysed in all samples. Frequency comparisons of -590C/T and stratified analysis including both cited SNPs were performed using chi-square tests. The -590C/T IL4 SNP was not found associated with T1D or RA when individual analyses were performed. However, a significant association with T1D emerged after stratification by L503F [p=0.02, odds ratio=1.95, 95% CI=1.07-3.55]. The location of the IL4 gene in the complex 5q31-33 genetic region, which contains many genes involved in immunological responses and presents linkage disequilibrium extended along many kilobases, makes necessary to interpret cautiously the previous IL4-association studies.     

Another explanation for the low allergy rate in the rural Alpine foothills

Background

IL4/IL4RA pathway plays an important role in atopy and asthma. Different polymorphisms in IL4 and IL4RA genes have been described. Particularly, -33C>TIL4 and 576Q>RIL4RA SNPs have been independently associated to atopy and asthma. The purpose of this study was to analyse these polymorphisms in a population of patients with a well-characterized asthma phenotype.

Methods

A total of 212 unrelated Caucasian individuals, 133 patients with asthma and 79 healthy subjects without symptoms or history of asthma or atopy and with negative skin prick tests were recruited. Lung function was measured by spirometry and asthma was specialist physician-diagnosed according to the ATS (American Thoracic Society) criteria and classified following the GINA (Global Initiative for Asthma) guidelines. Skin prick tests were performed according to EAACI recommendations. -33C>TIL4 was studied with TaqMan assay and 576Q>RIL4RA by PCR-RFLP technique. Hardy-Weinberg equilibrium was analysed in all groups. Dichotomous variables were analysed using χ², Fisher exact test, Monte Carlo simulation test and odds ratio test. To model the effects of multiple covariates logistic regression was used.

Results

No statistically significant differences between the group of patients with asthma and the controls were found when the allele and genotype distribution of -33C>TIL4 and 576Q>RIL4RA polymorphisms were compared. However, the T allele of the -33C>TIL4 SNP was more frequent in patients with persistent asthma. Multivariate analysis adjusted for age and sex confirmed that carriers of allele T had an increased risk of persistent asthma (OR:2.77, 95%CI:1.18–6.49; p = 0.019). Analysis of combination of polymorphisms showed that patients carrying both the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA had an increased risk of asthma. This association was particularly observed in persistent asthma [Fisher's p value = 0.0021, Monte Carlo p value (after 10⁴ simulations) = 0.0016, OR:3.39; 95% CI:1.50–7.66].

Conclusion

Our results show a trend of association between the genetic combination of the T allele of -33C>TIL4 and the A allele of 576Q>RIL4RA with asthma. This genetic variant was more frequently observed in patients with persistent asthma. As long as this study was performed in a small population, further studies in other populations are needed to confirm these results.     

Evidence for association and linkage between atopy, airway hyper-responsiveness, and the β subunit Glu237Gly variant of the high-affinity receptor for immunoglobulin E in the French-Canadian population

Following detection of linkage between atopy and chromosome 11q13 markers, association between this disorder and variants of the beta subunit of the high-affinity receptor for immunoglobulin E (FcepsilonRI-beta, a candidate gene for asthma-related conditions co-localizing within the same region) was reported in Australian, British and Japanese populations. Investigations in several other ethnic groups failed to replicate these observations. Due to the complexity of defining intermediate phenotypes related to asthma, detection of such associations may have been hampered by clinical misclassifications. To assess whether the FcepsilonRI-beta gene was involved in atopy and/or airway hyperresponsiveness (AHR) in the French-Canadian population, we conducted a case-control study in 200 subjects using strict criteria for asthma and related conditions. The Ile181Leu and Glu237Gly FcepsilonRI-beta sequence variants were tested exploiting two amplification refractory mutation systems. No association was detected between atopy or AHR and the Ile181Leu FcepsilonRI-beta variant. However, a strong association was observed between atopy and the Glu237Gly FcepsilonRI-beta variant (odds ratio=12.25). Four large Eastern Québec families (n=106 subjects) were also recruited to perform a genetic linkage study. We observed suggestive evidence of linkage between atopy and the Glu237Gly FcepsilonRI-beta variant (Zmax=2.30). This study is the first to detect the presence of an association between atopy and the Glu237Gly FcepsilonRI-beta variant in French-Canadians. Our data suggest that a susceptibility locus for atopy is located on chromosome 11q13 in this population.     

The CTLA-4 +49 A/G and ?318 C/T polymorphisms and susceptibility to asthma: a meta-analysis

The aim of this study was to explore whether cytotoxic T lymphocyte antigen-4 (CTLA-4) +49 A/G, and -318 C/T polymorphisms confer susceptibility to asthma. A meta-analysis was conducted on the associations between the CTLA-4 +49 A/G, and -318 C/T polymorphisms and asthma using; (1) allele contrast, (2) the recessive model, (3) the dominant model, and (4) the additive model. Eight studies on the CTLA-4 polymorphisms and asthma involving 2,330 patients with asthma and 1,743 control subjects were included in this meta-analysis. The meta-analysis revealed an association between asthma and the CTLA-4 +49 A/G polymorphism under the dominant model in Asians (OR = 0.758, 95 % CI = 0.599-0.958, p = 0.020). Stratification by age indicated an association between the CTLA-4 +49 GG+GA genotype and asthma in children (OR = 0.690, 95 % CI = 0.497-0.957, p = 0.026), but not in adults (OR = 0.837, 95 % CI = 0.598-1.172, p = 0.300). Furthermore, stratification by atopy status indicated an association between the CTLA-4 +49 G allele and atopic asthma (OR = 0.639, 95 % CI = 0.464-0.881, p = 0.006), but not non-atopic asthma (OR = 0.706, 95 % CI = 0.385-1.294, p = 0.266). There was no association between asthma and the CTLA-4 -318 C/T polymorphism for the whole population, or when stratified by ethnicity, age, or atopy status. This meta-analysis demonstrates that the CTLA-4 +49 A/G polymorphism confers susceptibility to asthma in Asians, children, patients with atopy status, but there was no association between the CTLA-4 -318 C/T polymorphism and asthma susceptibility.     

V75R576 IL-4 receptor alpha is associated with allergic asthma and enhanced IL-4 receptor function

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.     

A major susceptibility gene for asthma maps to chromosome 14q24

Asthma is a complex genetic disorder with a heterogeneous phenotype, largely attributed to the interactions among many genes and between these genes and the environment. Numerous loci and candidate genes have been reported to show linkage and association to asthma and atopy. Although some studies reporting these observations are compelling, no gene has been mapped that confers a sufficiently high risk of asthma to meet the stringent criteria for genomewide significance. Using 175 extended Icelandic families that included 596 patients with asthma, we performed a genomewide scan with 976 microsatellite markers. The families were identified by cross-matching a list of patients with asthma from the Department of Allergy/Pulmonary Medicine of the National University Hospital of Iceland with a genealogy database of the entire Icelandic nation. We detected linkage of asthma to chromosome 14q24, with an allele-sharing LOD score of 2.66. After we increased the marker density within the locus to an average of one microsatellite every 0.2 cM, the LOD score rose to 4.00. We designate this locus "asthma locus one" (AS1). Taken together, these results provide evidence of a novel susceptibility gene for asthma on chromosome 14q24.     

Resequencing candidate genes implicates rare variants in asthma susceptibility

Common variation in over 100 genes has been implicated in the risk of developing asthma, but the contribution of rare variants to asthma susceptibility remains largely unexplored. We selected nine genes that showed the strongest signatures of weak purifying selection from among 53 candidate asthma-associated genes, and we sequenced the coding exons and flanking noncoding regions in 450 asthmatic cases and 515 nonasthmatic controls. We observed an overall excess of p values <0.05 (p = 0.02), and rare variants in four genes (AGT, DPP10, IKBKAP, and IL12RB1) contributed to asthma susceptibility among African Americans. Rare variants in IL12RB1 were also associated with asthma susceptibility among European Americans, despite the fact that the majority of rare variants in IL12RB1 were specific to either one of the populations. The combined evidence of association with rare noncoding variants in IL12RB1 remained significant (p = 3.7 × 10(-4)) after correcting for multiple testing. Overall, the contribution of rare variants to asthma susceptibility was predominantly due to noncoding variants in sequences flanking the exons, although nonsynonymous rare variants in DPP10 and in IL12RB1 were associated with asthma in African Americans and European Americans, respectively. This study provides evidence that rare variants contribute to asthma susceptibility. Additional studies are required for testing whether prioritizing genes for resequencing on the basis of signatures of purifying selection is an efficient means of identifying novel rare variants that contribute to complex disease.     

The gene encoding interleukin-13: a susceptibility locus for asthma and related traits

Asthma is a complex inflammatory disorder controlled by both genetic and environmental influences. Multiple genetic analyses have identified the T helper type 2 (Th2) cytokine gene cluster on chromosome 5q as a susceptibility locus for asthma. Recently, the Th2 cytokine interleukin-13 has been shown to be a critical mediator of the asthma phenotype in murine models. In this commentary we discuss several recent studies that have identified polymorphisms in the gene encoding interleukin-13. The consistent genetic associations of interleukin-13 with asthma and related traits across diverse ethnic populations in these studies provides strong support for the candidacy of this cytokine as a susceptibility locus for asthma and atopy on chromosome 5q31.     

IL2RA Genetic Heterogeneity in Multiple Sclerosis and Type 1 Diabetes Susceptibility and Soluble Interleukin-2 Receptor Production

Multiple sclerosis (MS) and type 1 diabetes (T1D) are organ-specific autoimmune disorders with significant heritability, part of which is conferred by shared alleles. For decades, the Human Leukocyte Antigen (HLA) complex was the only known susceptibility locus for both T1D and MS, but loci outside the HLA complex harboring risk alleles have been discovered and fully replicated. A genome-wide association scan for MS risk genes and candidate gene association studies have previously described the IL2RA gene region as a shared autoimmune locus. In order to investigate whether autoimmunity risk at IL2RA was due to distinct or shared alleles, we performed a genetic association study of three IL2RA variants in a DNA collection of up to 9,407 healthy controls, 2,420 MS, and 6,425 T1D subjects as well as 1,303 MS parent/child trios. Here, we report “allelic heterogeneity” at the IL2RA region between MS and T1D. We observe an allele associated with susceptibility to one disease and risk to the other, an allele that confers susceptibility to both diseases, and an allele that may only confer susceptibility to T1D. In addition, we tested the levels of soluble interleukin-2 receptor (sIL-2RA) in the serum from up to 69 healthy control subjects, 285 MS, and 1,317 T1D subjects. We demonstrate that multiple variants independently correlate with sIL-2RA levels.     

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.     

Polymorphism of interleukins and interleukin receptor genes: population distribution and association with atopic bronchial asthma

Population distribution and pathogenetic significance for bronchial asthma (BA) of the eight polymorphic variants of six interleukin--(IL) and interleukin receptor genes, C-589T, G/C 3'-UTR IL4, C-703T IL5, T113M IL9, Q551R, 150V IL4RA, and G1972A IL5RB, was examined. In the population samples of Russians, Tajiks, Buryats, and Tuvinians racial and ethnic specificity of these polymorphisms was established. These specific features were manifested as population-specific "enetic portraits" in respect of polymorphic allele frequencies. Analysis of the BA patients and their relatives from Tomsk by use of transmission/disequilibrium test (TDT) revealed the presence of a statistically significant association between the C-703 IL5 allele and the disease (P = 0.005). This is the first evidence of an association between the IL5 gene polymorphism and BA.     

Unifying candidate gene and GWAS Approaches in Asthma

The first genome wide association study (GWAS) for childhood asthma identified a novel major susceptibility locus on chromosome 17q21 harboring the ORMDL3 gene, but the role of previous asthma candidate genes was not specifically analyzed in this GWAS. We systematically identified 89 SNPs in 14 candidate genes previously associated with asthma in >3 independent study populations. We re-genotyped 39 SNPs in these genes not covered by GWAS performed in 703 asthmatics and 658 reference children. Genotyping data were compared to imputation data derived from Illumina HumanHap300 chip genotyping. Results were combined to analyze 566 SNPs covering all 14 candidate gene loci. Genotyped polymorphisms in ADAM33, GSTP1 and VDR showed effects with p-values <0.0035 (corrected for multiple testing). Combining genotyping and imputation, polymorphisms in DPP10, EDN1, IL12B, IL13, IL4, IL4R and TNF showed associations at a significance level between p = 0.05 and p = 0.0035. These data indicate that (a) GWAS coverage is insufficient for many asthma candidate genes, (b) imputation based on these data is reliable but incomplete, and (c) SNPs in three previously identified asthma candidate genes replicate in our GWAS population with significance after correction for multiple testing in 14 genes.     

Molecular screening and association study of IL15 gene polymorphisms in rheumatoid arthritis

Interleukin 15 (IL-15) is a pleiotropic pro-inflammatory cytokine known to play a relevant role in rheumatoid arthritis (RA) pathogenesis. In this study we aimed to investigate for the first time the contribution of IL15 gene to RA susceptibility. We screened 13 single nucleotide polymorphisms (SNPs) localised within IL15 regulatory regions (promoter, 5' UTR region and 3' UTR region) in a total of 420 individuals, who were genotyped by direct sequencing of PCR products. In addition, an association study of these IL15 SNPs was conducted in three independent case-control cohorts of Spanish Caucasian origin, including a total of 645 RA patients and 656 healthy controls. The presence of the 13 selected IL15 SNPs in our population was confirmed and no new genetic variants were found. The distribution of the IL15 selected SNPs in RA patients and controls showed no statistically significant deviation in any of the populations studied. Additionally, we performed a haplotype analysis that revealed three IL15 haplotype blocks. None of the haplotype blocks was associated with RA susceptibility or severity in the three cohorts analysed. Our results suggest that the IL15 gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.     

哮喘的分子遗传学研究进展

哮喘病和易感症的遣传学病因是复杂的,现可通过候选基因和易感位点筛选技术来进行研究。本文综述了哮喘易感基因的研究进展,同时讨论了哮喘遗传学研究对哮喘临床的应用前景。 Abstract:The genetics of asthma and atopy is complex,but can be approached by studies of both candidate genes and mapping of susceptibility loci.The progress in susceptibility genes for asthma and atopy is reviewed.New therapeutic approaches to asthma which are based on the study on asthma genetics for future are summarized.     

Significant evidence for linkage of mite-sensitive childhood asthma to chromosome 5q31-q33 near the interleukin 12 B locus by a genome-wide search in Japanese families

Childhood-onset asthma is frequently found in association with atopy. Although asthmatic children may develop IgE antibodies against variety of allergens, asthma is associated primarily with allergy to house-dust mites, molds, or other allergens. In this study, we conducted a genome-wide linkage search in 47 Japanese families (197 members) with more than two mite-sensitive atopic asthmatics (65 affected sib-pairs) using 398 markers. Multipoint linkage analysis was carried out for atopic asthma as a qualitative trait using the MAPMAKER/SIB program. We observed significant evidence for linkage with maximum lod scores (MLS) of 4.8 near the interleukin 12 B gene locus on chromosome 5q31-q33. In addition, suggestive evidence on 4q35 with MLS = 2.7 and on 13q11 with MLS = 2.4 was obtained. The other possible linkage regions included 6p22-p21.3 (MLS = 2.1), 12q21-q23 (MLS = 1.9), and 13q14.1-q14.3 (MLS = 2.0). Many of the linkage loci suggested in this study were at or close to those suggested by genome-wide studies for asthma in Caucasian populations. The present study suggests the contribution of the interleukin 12 B gene or nearby gene(s) to mite-sensitive atopic asthma and a considerable number of genetic variants common across Caucasians and Japanese populations contributing to asthma, although the relative importance of various variants may differ between the groups.