(Chest. 2003;123:363S-368S.)
© 2003
American College of Chest Physicians
Mapping Susceptibility Genes for Allergic Diseases*
Timothy D. Howard, PhD;
Deborah A. Meyers, PhD and
Eugene R. Bleecker, MD, FCCP
* From the Department of Pediatrics (Drs. Howard and Meyers), Center for Human Genomics; and Department of Medicine (Dr. Bleecker), Center for Human Genomics, Wake Forest University Health Sciences, Winston-Salem, NC.
Correspondence to: Eugene R. Bleecker, MD, FCCP, Center for Human Genomics, Wake Forest University Health Sciences, Medical Center Blvd, Winston-Salem, NC 27157; e-mail: ebleeck{at}wfubmc.edu
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Abstract
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Allergic diseases are most likely due to interactions between genetic and environmental factors. While many of the environmental components have been studied for years, only recently has significant progress been made in identifying the genes responsible for susceptibility or expression of these diseases. Genome-wide screens in various populations have identified the locations of susceptibility genes for asthma and atopy, as well as associated phenotypes such as bronchial hyperresponsiveness and increased total serum IgE levels. In addition, this positional cloning approach has led to the discovery of several genes for asthma or related phenotypes, which is extending our understanding of the pathophysiology of allergic diseases. As these genes are identified and characterized, the relationships of these genes to each other and the environment will become important areas of research. Understanding the basic interactions that lead to the development of allergy and asthma will lead to new therapeutic approaches that will be used to modify the development and clinical progression of these common disorders.
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Introduction
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In the last 10 years, there has been significant progress made in delineating the role of genetic susceptibility in the expression and development of asthma and atopy. Asthma is an inflammatory airways disease caused by a complex interaction between host susceptibility (genetics) and diverse environmental factors. It is apparent from ongoing research that no single susceptibility gene confers a major risk for this disease. More likely, multiple genes acting either alone or in combination with other genes increase the risk for asthma and/or other atopic conditions after exposure to a diverse group of environmental triggers. Thus, it is possible that different combinations of asthma and allergy susceptibility genes interacting with varying external factors will regulate the onset or expression (severity) of asthma (Fig 1
). A comprehensive standardized approach to characterizing phenotypic variation and heterogeneity in asthma is necessary to identify genes that regulate the expression and progression of asthma and closely associated allergic traits.
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Positional Cloning of Susceptibility Genes
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Positional cloning, a method in which families are used to map genes to chromosomal regions by examining co-segregation between the disease phenotype and DNA markers, has been very effective in identifying disease genes for Mendelian (single gene) genetic diseases. This approach is now proving to be increasingly useful in asthma (as recently shown by the identification of the ADAM33 gene on chromosome 20),1
as well as in other complex human diseases (eg, Crohn disease).2
While this approach is useful in common diseases, there are inherent difficulties since disease susceptibility is due to multiple genes and their interactions with environmental factors. The use of well-characterized and diverse populations or ethnic groups to evaluate the role of proposed asthma genes is necessary to understand the specific role of each gene, as well as gene-gene and gene-environment interactions in the development of asthma. More homogeneous populations such as the Dutch or Hutterites may provide a better opportunity to use genetic analytic techniques that include linkage disequilibrium and haplotype analysis to identify asthma susceptibility genes.3
4
5
6
Since asthma and allergy are common diseases, understanding the interaction between genes and the environment has important public health implications. A goal of genetic studies is to provide further insight into disease pathophysiology, and ultimately lead to new and more effective therapeutic interventions, new diagnostic methods for presymptomatic diagnosis, the development of strategies for disease prevention in susceptible individuals, and delineation of the interaction between genotype and the response to specific treatments (pharmacogenetics).
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Characteristics of Asthma and Allergy Phenotypes
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To investigate the genetic basis of a disease such as asthma, it is useful to evaluate intermediate, associated phenotypes such as bronchial hyperresponsiveness (BHR) and total serum IgE levels that can be measured objectively in all individuals in a family. BHR is associated with asthma and atopy, as observed by the close association of serum IgE levels, BHR, and asthma in asymptomatic children.4
7
This relationship illustrates how closely these factors are related in the pathogenesis and expression of asthma. Therefore, it is appropriate to use BHR and total serum IgE levels as relevant associated phenotypes, as well as other measures of the allergic phenotype such as total serum eosinophil counts, skin test responses to common allergens, and specific IgE measures.
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Genome-Wide Screens in Allergy and Asthma
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In the last 5 years, multiple studies have been performed in individuals from Australia, The United States, Germany, France, The Netherlands, China, and the United Kingdom4
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These studies have contributed significantly to the mapping of allergy and asthma genes by conducting genome-wide screens in different human populations. These approaches use linkage analysis to identify regions of the genome that are statistically linked to a specific phenotype. Table 1
lists genetic term definitions. Results of linkage analysis are usually expressed as a lod score (log of the odds) for a trait to be linked to a specific chromosomal region. One example of this method is the genome-wide screen performed in Hispanic families from New Mexico11
(Fig 2
). Regions of interest observed in the Hispanic population include chromosomes 1p, 2q, 5q, 11p, 12q, 14q, 17q, and 21q. Authors from the Collaborative Study on the Genetics of Asthma (CSGA)11
also demonstrated evidence for gene-gene interactions using analytic techniques showing interrelationships between different chromosomal regions (Fig 3
). These data support our hypothesis that several genes interact in the development of asthma.
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Figure 2.. Genome-wide screen results for the asthma phenotype in the Hispanic population. The lod score is shown on the y-axis. Data from all 22 autosomal chromosomes are shown simultaneously on the x-axis.
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Figure 3.. Genetic interactions determined from the genome-wide screen in the CSGA populations. Evidence for linkage on chromosomes 12q and 8p increased when conditioned on chromosome 11q. Likewise, conditioning on chromosome 14q increased the evidence for linkage on chromosomes 12q and 15q and conditioning on 1p increased the evidence for linkage on 8p, 15q and 5q.
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Results of other linkage studies showing evidence for linkage to multiple chromosomal regions are summarized in the Asthma and Allergy Gene Database (http://cooke. gsf.de/asthmagen).16
Instead of reviewing all of these studies, two examples will be presented to demonstrate results from these studies that will lead to an improved understanding of the pathogenesis and progression of asthma and allergic disorders.
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Examples of Positional Cloning of Asthma Susceptibility Genes
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Chromosome 5q
Linkage to chromosome 5q was reported initially using a candidate gene approach, due to the multiple genes on 5q that may be important in the regulation of IgE and the development or progression of inflammation associated with allergy and asthma. Linkage to 5q has been observed in several populations for a range of phenotypes ranging from asthma and BHR to total serum IgE levels.
In the inbred and genetically isolated Amish population, linkage to 5q in the region of several of the cytokines was observed for regulation of total serum IgE levels.17
In a Dutch asthma population, strong evidence of linkage was found for total serum IgE variation and susceptibility to BHR.18
19
Evidence for linkage to a region distal to the cytokine region on 5q was observed in the Hutterites for the "loose" definition of asthma that included subjects with asthma, BHR, or asthmatic symptoms.5
In the CSGA Hispanic families, evidence for linkage to this region of 5q was observed for the asthma phenotype.11
In addition, evidence for gene-gene interaction was observed in the CSGA family study showing an interactive effect between genes on 5q and 1p (Fig 3)
.11
It appears that there may be linkage to at least two separate regions on 5q, the cytokine region, which includes interleukin (IL)-4 and IL-13, and a more distal region.
An association study was performed by comparing the frequency of sequence variants in IL13 in unrelated individuals (Dutch probands and spouses) with and without the disease phenotype.20
First, IL13 was sequenced in 20 probands and 20 unaffected spouses to detect sequence variants. Ten single-nucleotide polymorphisms (SNPs), including four novel ones, were detected. A subset of SNPs was then genotyped in 184 probands and their spouses. Significant evidence for association was observed for two phenotypes (both of which showed evidence for linkage to this region) with a SNP in the IL13 promoter region: asthma (p = 0.008) and BHR (p = 0.007). Significant evidence was also observed for skin test responsiveness but not to total serum IgE levels, even though there is evidence of linkage for variation in total serum IgE levels to this chromosomal region. A previous study21
of allergic asthma also found an association with the promoter SNP. No significant associations were observed for the Arg130Gln SNP at position 4257, although three previous studies22
23
24
found associations with asthma-related phenotypes including total serum IgE levels.
IL4 and IL13 are both located on chromosome 5q and share a common receptor component, IL-4R-
. Therefore, IL13 and IL4RA were studied for evidence of a gene-gene interaction in relation to asthma and allergy phenotypes in the Dutch population.25
Significant evidence for an association was observed for several SNPs in IL4RA, including an association with total serum IgE levels (S478P, p = 0.0007). Together, this SNP in IL4RA (S478P) and the - 1111 promoter variation in IL13 shown to be associated with BHR showed evidence for a significant gene-gene interaction. Individuals with the high-risk genotype for both genes had an approximate fivefold higher risk for developing asthma than those with both nonrisk genotypes (p = 0.0004). Thus, it may be possible to assess risk for the development of asthma by evaluating SNPs in several genes in an individual.
Chromosome 2q
A second example of the positional cloning approach involves mapping total serum IgE levels in the same Dutch population. A consistent region showing evidence for linkage to total serum IgE or other allergy phenotypes is chromosome 2q32-q33, which has been identified in our Dutch asthma family study,4
14
and also in other populations.6
12
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CTLA-4 is a candidate gene in the 2q32-q33 region and is involved in T-cell activation and therefore may be important in IgE regulation and susceptibility to immunologic diseases. Three polymorphisms have been studied in immune disorders: a C/T polymorphism at position – 319 of the promoter,26
an A/G change at position + 49 of exon 1 (encodes for an amino acid change of Thr to Ala),27
and a microsatellite AT repeat in the 3' untranslated region (UTR).28
CTLA-4 has been examined in asthma and allergy phenotypes with mixed results. No association was observed in a sample of German individuals ascertained randomly or through an individual with atopy,29
or in a Japanese group of atopic asthma subjects, where the – 319, + 49, and 3'UTR polymorphisms were examined.30
In a separate case-control study comparing patients with asthma to healthy control subjects, no association with the – 319 or + 49 SNPs in CTLA-4 was observed with asthma, but an association with – 319 and total serum IgE levels was reported in patients with asthma.31
To test this gene for association with total serum IgE levels in the Dutch population, we first sequenced the gene in 96 individuals from Dutch, US white, African American, and Hispanic populations.32
Sequencing of the immediate putative 5' promoter regions and all four exons revealed four novel SNPs in CTLA-4 in addition to the four previously described polymorphisms. The probands and spouses from the 200 Dutch families were analyzed for five SNPs and the 3' UTR microsatellite in CTLA-4. Total serum IgE levels were significantly associated with the + 49 A/G and 3' UTR polymorphisms (p = 0.0005 and 0.006, respectively) [Fig 4 ]. Significant association was observed for the – 1147 C/T SNP with asthma and BHR (p = 0.012 and 0.008, respectively), but not with the allergy-related phenotypes. In addition, promoter analysis revealed a twofold decrease in transcription of the T-allele genetic variant, suggesting a decrease in CTLA-4 expression. This decrease would lead to less of the CTLA-4 receptor on the membrane, potentially extending the time of T-cell activation. These data suggest that the co-stimulatory pathway, and specifically CTLA-4, is important in the regulation of total serum IgE levels and the development of atopy and asthma.
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Figure 4.. Association of total serum IgE levels with the exon 1 (+ 49 A/G) and the 3' UTR (86/xx) polymorphisms of CTLA-4. Individuals with the AA and 86/86 genotypes had increased mean total serum IgE levels compared to individuals with the other genotypes.
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Summary
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Genetic approaches are useful in dissecting the complex genetic relationships between susceptibility to BHR, allergic disorders, and asthma. It is a major challenge to identify the genes responsible for susceptibility to complex multifactorial diseases such as asthma and BHR.33
Significant progress has been made in the last decade. Several regions of the genome have been identified where it is very likely that there are genes that confer increased susceptibility to asthma and BHR. As these genes are delineated, determining the specific role of each gene in the development of asthma as well as characterizing gene-environment correlations are important areas of research. By understanding the basic genetic mechanisms that lead to the development of allergy and asthma, new therapeutic interventions will be developed that will be used to modify the development and clinical progression of these common disorders.
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Footnotes
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Abbreviations: BHR = bronchial hyperresponsiveness; CSGA = Collaborative Study on the Genetics of Asthma; IL = interleukin; SNP = single-nucleotide polymorphism; UTR = untranslated region
This work was supported by the Netherlands Asthma Foundation grant AF 95.09 and National Institutes of Health grants R01HL/48341, R01HL/66393, and U01HL/49602.
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References
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Abstract
Introduction
