Atopy and asthma genes--where do we stand?

Abstract

Human beings are complex organisms living in a complex environment. It is not surprising, therefore, that many of the common diseases – such as asthma and other allergic diseases – which afflict human beings, are complex. While they may demand a high level of medical attention (usually because of mortality at an early age), the diseases which can be attributed to single, major genes are relatively rare. In contrast, the more common human diseases (>1% prevalence) typically exhibit genetic heterogeneity, appear to be under multifactorial control (where both genes and environmental factors control risk), and possibly exhibit both features. The inflammatory response has evolved because of its importance in resisting infection; however, its function is misdirected in allergic diseases, especially asthma. While the ensemble of genes within this system is diverse, it is replete with redundancies; for example, there are multiple genes encoding the component cytokines or cell-adhesion molecules and their receptors (1–3). The inflammatory system (like all complex subsystems that play vital roles in host survival) has proved to be very resistant to change in the Mammalia, despite the disparity in DNA sequence between species. Thus, many mutations can be tolerated within the genes of this system across different species, and the same is likely to be true (to a lesser extent) within a species. That is not to say that mutations have no effect, but that their effects are modulated by the interactive nature of the complex system. Our assumption, a priori, is that there will be multiple variants in multiple genes controlling the inflammatory response (including regulatory elements of these genes), and that these variants will have variable and interdependent effects on the expression of the different allergic disease phenotypes. Expression of these “inflammation genes” will also vary according to the environmental context. In view of these considerations, it is not surprising that genetic studies of the allergic diseases have met with limited success. Borecki et al. (4) attempted to relate the genetics of IgE regulation to the different manifestations of allergic disease using total IgE and clinical data from the 173 families studied earlier by Gerrard et al. (5). They showed that a major locus determining high IgE levels markedly influences the development of different manifestations of atopic disease. Specifically, when only asthma and atopic dermatitis were considered, the effect of the major gene was compatible with recessive inheritance. Meyers et al. (6) also have proposed a major autosomal recessive locus determining high IgE levels, whereas Martinez et al. (7) observed codominant inheritance of a major autosomal locus for this same trait. Cookson & Hopkin (8) also proposed autosomal dominant inheritance of atopy, and in a subsequent genetic linkage analysis (9) provided evidence that an “atopy gene” was linked to the D11S97 marker in chromosome 11q13. However, using a well-designed, complex segregation analysis on a random cohort of 131 nuclear families, Lawrence et al. (10) concluded that a major gene effect for any of the associated traits examined (wheeze, asthma, BHR [bronchial hyperresponsiveness], total IgE, hay fever, atopy, positive skin prick tests, and eczema) was unlikely. In one of his last papers before his untimely death, David Marsh (11) argued for reconsideration of the “reductionistic” approach traditionally used in analyzing complex phenomena such as atopic disease. As he described, even linkage analysis is a reductionistic strategy for detecting disease genes, although it remains one of the best tools that we have for the genetic dissection of complex traits. Marsh contended that, even though reductionism is a useful paradigm for deciphering complex systems, we need to consider its limitations. The benefits and potential pitfalls of reductionism can be best illustrated by considering David Marsh's own historic work: that is, studies of HLA-linked, specific immune responsiveness to “simple” allergens. Marsh and his colleagues focused on the HLA-D subregions (DR, DQ, and DP) of the human MHC to examine immune responsiveness to two of the major allergens of short ragweed, Amb a 5 (5 kDa) and Amb a 6 (9.9 kDa) (12–14). They postulated that immune recognition involved the interaction of a single MHC class II molecule (or a group of closely related class II molecules) with a single major T-cell epitope of the antigen. From this assumption, it was expected that unrelated individuals would share HLA-D genes in the cognate response to antigens. In fact, the group found a class II restriction limited to individuals carrying HLA-DR2 and Dw2 (DR2.2) in all but two of approximately 80 Caucasian IgE responders to Amb a 5. However, in atopic individuals who did not respond to Amb a 5, the DR2.2 frequency was similar to the Caucasian population at large (∼22%). The group went on to identify the actual class II restriction elements associated with the DR2.2 and DR2.12 phenotypes in Caucasians (Drαβ1*1501 molecule) and Asians (DRαβ1*1502 molecule), respectively. These studies clearly showed a cause-and-effect relationship between the presence of a particular HLA-D phenotype and specific IgE and IgG Ab responsiveness. Numerous studies confirmed Marsh's findings, and HLA-D associations were extended to giant ragweed, perennial rye, and even the mite allergens, Der p 1 and Der p 2 (15–21). However, it appears that with increasingly “complex” allergens (e.g., increasing numbers of T-cell epitopes), the associations between specific HLA alleles and immune responsiveness may be diluted. Furthermore, there seems to be a “dosage threshold” whereby an inverse relationship exists between higher allergen doses and the strength of the HLA association. For example, and as discussed by Marsh (11), the association between HLA-DR3 and immune responsiveness to the ryegrass allergen Lol p 3 was observed only among low total serum IgE responders; HLA-DR3 was not more frequent among Lol p 3 responders with high total serum IgE levels than Lol p 3 nonresponders with similarly high total serum IgE levels (22). These findings suggest that HLA-D specificity and another associated trait of atopy – total serum IgE – interact in the expression of specific immunoresponsiveness. Conversely, a different trend is observed with Der p 1 as the model of specific immunoresponsiveness when we consider the evidence presented by Hizawa et al. (23) for linkage of Der p 1-specific IgE responsiveness and chromosome 6p21 markers in the HLA-D region among a group of 45 Caucasian families (P=0.0064). Evidence for linkage to the HLA-D region was not observed among a group of 53 African-American families, despite the fact that all families, Caucasian and African-American, were recruited as part of the same study (the USA-based, NHLBI-funded Colla-borative Study on the Genetics of Asthma [CSGA]). In both groups, mean log(total IgE) was significantly higher among the Der p responders, suggesting that the consideration of interacting factors beyond total serum IgE is necessary to understand the pathology of specific immunoresponsiveness in these two groups of patients. The search for genes contributing to the interrelated diseases of atopy and asthma has been further confounded by another factor: definition of the phenotype. There is no quantitative “marker” of asthma per se, and no “gold standard” definition against which to validate the diagnostic usefulness of physiologic measures (e.g., BHR) typically assessed when characterizing an individual as either “asthmatic” or “nonasthmatic” (24). This feature is somewhat (although not entirely) unique to asthma, as certain other complex diseases can be defined by quantitative traits, or a series of measurable “biomarkers”, such as prostate-specific antigen (PSA) in prostate cancer, fasting insulin and C-peptide levels in non-insulin-dependent diabetes mellitus (NIDDM [25]), or spinal bone-mineral density, spinal Z(BMD) in osteoporosis (26), each of which has proved useful in teasing out susceptibility genes. Lastly, asthma is a “moving target”, a disease quite often episodic in nature, where the natural history can change substantially during the course of an individual lifetime. Although asthma is characterized by both acute and chronic airway inflammation (most typically, it is an IgE-mediated, or “allergic,” inflammation in response to aeroallergens), the symptoms observed to result from the increased smooth-muscle contraction, mucus hypersecretion, and vascular permeability following acute inflammation may not vary substantially from what is observed to follow structural and functional changes over time, as in the case of chronic airway inflammation. In advanced years of life, the diagnosis or confirmation of a diagnosis of asthma can also be confounded by other pulmonary diseases, such as chronic obstructive pulmonary disease (COPD), or certain effects of heavy smoking. To this end, analysts are frequently faced with the difficult task of “pigeon-holing” subjects of equivocal diagnosis into a definitive category (e.g., “asthmatic” or “normal”), or removing such subjects from the analysis altogether. Genetic mapping, the method by which the inheritance pattern of a trait is compared with the inheritance pattern of chromosomal regions, was revolutionized with the availability of highly polymorphic, genetic markers in the 1980s (27). These “flags” or “signposts”, which can indicate the approximate location of genes suspected of contributing to a particular disease, have aided in the isolation of polymorphisms, or allelic variants, that play a role in the expression of a particular phenotype or disease. Positional cloning is a process where linkage is established between the presence of the disease/disease-associated phenotype(s) and these highly polymorphic genetic markers, with increasing levels of precision leading to the isolation of a gene of interest without prior knowledge of its function. Just over a decade ago, linkage to atopy by polymorphic markers was reported for the first time (9). In the following 10 years, evidence of linkage to over 22 loci of 15 autosomal chromosomes and the pseudoautosomal region of Xq/Yq has been reported from numerous candidate gene studies (Table 1). Several genome-wide screens for asthma and/or atopy have been completed, with more underway, including collaborations with the biotechnology and pharmaceutic industries. Although it can be argued that the large number of candidate loci may reflect a type I error (false positive), especially in conventional case-control design studies, replication is reassuring; in fact, roughly half of the reported linkages shown in Table 1 have been replicated in one or more independent populations. Conclusions drawn from most of these various linkage studies are speculative at best, however. Each of the regions where significant and reproducible evidence of linkage has been identified contains multiple candidate genes. The best evidence for linkage in any one of these regions tends to extend over relatively large portions of the chromosome – up to 10 centimorgans (cM) or greater – rendering the pinpointing of any specific locus very difficult. This problem is confounded further by the limitations of the methodology: statistical approaches commonly used for linkage analysis of genotype data generated by microsatellite markers, even in the case of fine mapping with the aim of localizing a gene to a region of <1 cM (1 cM=about 1 million base pairs), do not usually allow one to pinpoint the exact gene of interest per se, but instead narrow the region of peak linkage score, which still may include hundreds of genes. Furthermore, few studies to date have achieved “significant” statistical evidence for linkage as defined by the estimate of P values ≤2×10−5, or a LOD of 3.6 for genome-wide scans (28). It has been suggested that, by using such strict criteria for linkage analysis, we may miss true linkages for complex diseases (29, 30); in fact, a more relaxed criterion (e.g., P<1×10−2) might be more appropriate for initial screenings (31) in genome scans. As it is not uncommon to identify linkage of modest levels of significance to a complex trait, replication in independent populations becomes critical in validating regions that are “suggestive” of linkage. Comparisons can be further confounded, however, by differen-ces within populations themselves, or population heterogeneity, which includes both genotypic and environmental heterogeneity. For example, population stratification, in which two subgroups differ in both allele frequency and disease frequency, may erroneously suggest an allelic association between a genetic marker and a disease (32). Similarly, if some families segregate for one gene linked to a given marker, the evidence for linkage may be counteracted by other families segre-gating for another gene (this is called linkage heterogeneity). One approach to minimize the effect of linkage heterogeneity is to study families from populations that are genetically homogeneous, a trait which increases the likelihood that the phenotype will be influenced by the same locus (27, 33–35). Similarly, genetically homogeneous populations are less likely to have genetically distinct strata that create confounding in association studies. Because one really cannot be sure that a person has the atopy (or asthma) phenotype if that individual has never had sufficient exposure to certain triggers necessary to elicit the response, selecting populations for study in which allergen exposure is relatively uniform optimizes the search for atopy genes. For this reason, the focus in asthma genetics is increasingly shifting toward the dissection of gene–environment interactions, targeted primarily (but not exclusively) on the effect of conventional environmental exposures (e.g., indoor allergen exposure, diet, active/passive smoking) on the risk of disease for those individuals with a genetic propensity for disease (e.g., positive family history for asthma). If one models gene– environment interactions in founder populations where the environmental background is relatively uniform, susceptibility alleles will be more similar (36). Not surprisingly, some of the most consistent and reproducible evidence for linkage to asthma and/or atopy has been based on inbred populations of rela-tively homogeneous genetic and environmental backgrounds (e.g., Amish, Hutterites [North America]) and/or restricted populations who are homogeneous for both their environment and genetic background without measurable inbreeding (e.g., Japanese) (37–40). Notwithstanding the shortfalls of linkage studies conducted to date, it is useful to consider the loci for which the most convincing evidence for linkage has been shown over the past decade. The initial studies were based on a candidate gene approach and include reports of linkage to 5q, 6p, 11q, and 12q, which, interestingly, remain four of the most solid regions for asthma and atopy in terms of strength of evidence (e.g., magnitude of LOD scores) and replication among independent populations (Table 1). An in-depth reference to these and other studies is available on both http://cooke.gsf.deand http://panther.qimr.edu.au/davidD/asthma6.html. One of the earliest studies showing evidence for linkage to an “atopy” gene, research which has been widely replicated, is the work of Cookson's group at Oxford (9). After a prelim-inary screen using 17 polymorphic markers at various candidate genes, the group reported evidence for linkage of atopy to 11q13, and subsequently provided further evidence for linkage in two separate popu-lations (UK and Australia) as part of a genome-wide search (41). Although, initially, other studies were unable to replicate Cookson's findings (see discussion at Duffy's website http://panther.qimr.edu.au/davidD/asthma6.html), many groups have now confirmed linkage at this locus (40, 42–45). Soon after Cookson's initial findings, he proposed that the FCER1B gene, which encodes the β chain of the high-affinity receptor for IgE, is the primary candidate in this region (41, 46). Linkage at this locus has been shown for many of the phenotypes associated with atopy, including asthma (41), BHR (47), total and specific IgE (41, 48), and atopic dermatitis (49), with some of the highest LOD scores (e.g., 9.35 in Shirakawa et al. [40]) reported in linkage studies of asthma and atopy. One of the regions for which there is strong evidence of linkage to asthma and atopy (especially total serum IgE levels) is chromo-some 5q31–q33. This region contains multiple can-didate genes for allergy and asthma, including IL3, IL4, IL5, IL13, IL9, and CSF2 (GM-CSF), as well as the glucocorticoid receptor 1 (GRL1) and beta 2- adrenergic receptor (ADRB2), all of which play important interactive roles in the allergic response. Probably of greatest interest in this cluster is IL-4, important in immunoglobulin class-switching to IgE and in the differentiation of naive T cells into Th2/Th0 cells (50–53). Moreover, within 14 kB of IL-4 is the gene encoding IL-13, which has been shown to be an important mediator of an asthma-like phenotype in mice independent of IL-4 (54, 55). The first evidence for linkage of total IgE concentrations to the 5q31–q33 region was presented by Marsh et al. (37) and was based on 11 multiplex Amish families. Reports from the laboratories of Meyers et al. (6, 39), Rosenwasser et al. (56), Martinez et al. (57), and Holgate (58) are consistent on linkage to 5q31.1 and the IL4 region and associated phenotypes, including BHR and circulating eosinophils. Evidence for linkage of asthma and associated phenotypes to 5q31–q33 has, in fact, been replicated by many groups, including those using families from Japan (59), Australia (48), and the Hutterites, a genetic isolate (60). In addition to the potential importance of IL-4 as the major candidate in this 5q region, the IL-9 gene is also a likely candidate (58). IL-9 is a T-cell growth factor; it upregulates mast-cell proteases (mMCP-1, mMCP-2, and mMCP-4), granzyme B, and probably hematopoietic progenitors and B cells; and it activates STAT1, STAT3, and STAT5 (61–64). Recently, IL-9 was shown to induce lung eosinophilia in the mouse (65). Using the mouse model, Levitt's group found that BHR was linked specifically to the IL-9 locus, and supported their findings by determining that IL-9 expression was significantly reduced in hyporesponsive mice (66). The same group (67) went on to demonstrate evidence for linkage of asthma and BHR to markers at the IL-9 receptor, which has been localized in the 320-kb-long pseudoautosomal region of Xq/Yq (68). Despite the rather convincing evidence of linkage between a locus (or loci) in 5q31–q33 and asthma or atopy from over a dozen independent populations to date, there are other well-designed studies which did not detect evidence of linkage in this region (41, 69). Another region of interest for which replicative evidence for linkage to asthma and associated phenotypes by a candidate gene approach has been shown is on 12q15–q24.1 (70–74). This region contains candidate genes potentially important for asthma susceptibility including the gene encoding interferon-γ (IFNG), the cytokine which opposes the effects of IL-4. Other candidates include stem cell factor (SCF), insulin-like growth factor-1 (IGF1), the β subunit of nuclear factor-Y (NFYB), leukotriene A4 hydrolase (LTA4H), and B-cell translocation gene 1 (BTG1) (75). Also of interest in this region is STAT6, a gene important in the IL-4/IL-13-mediated signal transduction pathway. The first evidence for linkage between asthma and the associated phenotype “high total IgE” and markers in this region came from affected sib-pair and multipoint analyses of two diverse populations (71), the isolated, Caucasian Amish and multiplex Afro-Caribbean families from the West Indies island of Barbados. These findings have been supported by other groups, including the CSGA (76), Ober and colleagues (60), the Southampton group (74), and both the German Multicenter Atopy Study (MAS '90) (73) and the German Multicenter Allergy Study (31). Evidence for linkage has been observed over a very large distance (>60 cM) on 12q, and, for certain loci, linkage has been reported for both asthma and atopy; for others, the linkage was for asthma only. The genes involved in specific immunoresponsiveness can be divided into two types: genes that are directly involved in antigen recognition (e.g., HLAD, TCR) genes that are part of specific immunoresponsiveness independent of the precise antigenic specificity (e.g., TCR/CD3 complex signal-transduction, IL4 gene-regulation). Some of the earliest studies (prior to the use of polymorphic, microsatellite markers) on the genetics of atopy focused on the human leukocyte antigen (HLA)-D subregions (DR, DQ, and DP) of the human major histocompatibility complex (MHC) located on the short arm of chromosome 6 (6p21.3). Many of the initial studies were conducted by Marsh and his group (12–14), as already discussed. Linkage studies in this region have been less conclusive, particularly for the phenotype “asthma”. In a study of 20 families identified by an atopic asthmatic proband (allergic to mite) from Cartagena, Colombia, Caraballo & Hernandez (77) provided data suggesting the existence of HLA-linked genes controlling mite-specific IgE concentrations, and later determined that HLA-DR3 is associated with a response specific to Blomia tropicalis (a tropical storage mite). As part of the CSGA genome-wide screen, evidence for linkage between Der p 1-specific IgE responsiveness and chromosome 6p21 markers in the HLA-D region was found among a group of 45 Caucasian families (23). Using immunoblotting analysis to evaluate specific IgE immune responses to the Der p allergens, the group subsequently identified two alleles (the 196 base pair [bp] of D6S1281 and the 104 bp of DQCAR) that showed evidence of disequilibrium with a Der p-specific IgE response to a particular Der p polypeptide (78). Linkage between the phenotype “asthma” and this locus was also presented from the German genome-wide screen (31), but it is still not clear if both phenotypes are controlled by the same gene. Like HLAD, the T-cell receptor (TCR) is a molecule essential in foreign antigen recognition and handling. The majority of T-cell receptors are comprised of alpha and beta chains, and the genes encoding these products have been identified and are located on chromosomes 14 and 7, respectively. Using sib-pair analysis on two sets of families from the UK and Australia, Moffatt et al. (79) showed evidence for linkage of specific IgE responsiveness to the TcR-α/δ locus on chromosome 14q11. More recently, a Japanese group (80) found evidence for linkage of asthma to the TCR-β gene on chromosome 7q35, but did not find evidence for linkage to TCR-α. Linkages have been reported in a number of other regions, some of which have been replicated elsewhere and several that have yet to be confirmed (Table 1). A locus on chromosome 13q14.3–qter has been of particular interest in recent years, and evidence for linkage in this region has been shown for asthma (76, 81) and genetic regulation of total and specific immunoresponsiveness to Dermato-phagoides pteronyssinus (23, 41) in diverse pop-ulations. One candidate in this region is the recently localized tumor protein, translationally controlled 1 (TPT1), which encodes the human histamine-releasing factor (HRF) (82). Recently, Cookson's group des-cribed a physical map of the locus of interest and recovered additional microsatellite markers, further supporting that group's evidence for linkage to atopy and serum IgE levels on chromosome 13 (83). Another pathway of human immune regulation includes the immunoglobulin heavy chain G (IGHG). The gene encoding the constant portion of the heavy gamma 1, gamma 2, and gamma 3 chains is located on chromosome 14q32. A Swedish group (84) recently demonstrated that one allotype for each of the three IGHG genes (IGHG1[f/f], IGHG2[n/n], and IGHG3[b/b]) was in linkage disequilibrium with specific IgE responsiveness, whereas an alternative allotype (IGHG1[a/a], IGHG2[-n/-n], and IGHG3[g/g]) was in linkage disequilibrium with asthma, suggesting that different pathways for the different (but associated) phenotypes may be determined by different genetic mechanisms at the same locus. Another region rich in genes associated with asthma and airway inflammation, and one for which there is compelling evidence for linkage, is 17q11.2–q21, where the C-C chemokine-encoding genes are clustered (85, 86). Of particular interest in this cluster is RANTES, a potent chemoattractant for lymphocytes, monocytes, eosinophils, and basophils (73, 87, 88). Initial analyses from the CSGA, using multipoint nonparametric linkage methods, provided evidence for an asthma susceptibility locus on chromosome 17p11.1–q11.2 (MLod=1.90, P=0.0015) in African-American families (76). The genome-wide approach involves the scanning of the entire human genome with a large collection of genetic markers (∼300–400) typically spaced 10–20 cM apart. As with the candidate gene approach, the appropriate multipoint linkage statistic S(x) is calculated at each position x along the genome, and the score statistic S shows the deviation in IBD sharing from what would be expected by chance alone for pairs or larger sets of relatives. The benefit of conducting genome-wide scans is obvious: the odds of identifying susceptibility loci are considerably higher if one is looking throughout the genome rather than at a single candidate region. The disadvantage of conducting such a screen is the cost – multiplying ∼360 markers times the number of families necessary for identification of an unobserved disease gene(s). This can be prohibitive for a disease such as asthma, which has a “relative risk for siblings” (λs, or the risk that a sibling of an asthmatic subject also has asthma, divided by the risk that an unrelated individual from the same general population has asthma) of only about 2.0, as it has been suggested that the number of families required for linkage analysis exceeds 2500 (89). In fact, several groups have successfully completed genome-wide studies using families numbering in the hundreds, and this has resulted in some significant and reproducible findings. The first results of a genome-wide search for susceptibility loci was reported in 1996, and they showed evidence for linkage to asthma and associated phenotypes in five novel regions in families from Australia (41). The following year, the CSGA published findings from their genome-wide search based on families from three ethnic groups (Caucasian, African-American, and Hispanic [USA]) and reported linkage to six additional, novel regions (76). Interestingly, the combined genome-wide scans in the Cookson study and the CSGA showed linkage in only one common region (13q). Cookson's group did not find evidence of linkage for two of the five new loci in a second panel of families from the UK, nor did they find evidence of linkage to several previously identified regions (e.g., 5q). Similarly, the CSGA genome-wide search did not provide evidence of linkage in more than one ethnic group for any of the six novel regions. In 1998, Ober et al. published their findings on a genome-wide search for asthma in a large group (361) and a replicate group (292) of Hutterites, a religious isolate (colony) living in South Dakota (USA) (60). They found evidence for linkage in four regions previously reported and in one novel region, 3p24.2–p22 (60). More recently, the German Asthma Genetics Group reported replicative evidence for linkage on chromosomes 6p, 9q, and 12q, and novel evidence for linkage on chromosome 2pter in 97 families selected by two siblings with asthma (31). The Oxford group conducted a genome-wide screen in a cross between Biozzi BP2 and BALB/c mice, and identified evidence for linkage in four loci syntenically homologous to five human regions, including 11q23 near the gene encoding the IL-10 receptor (IL-10R) 12q22–q24 5q31 17q12–q22 6p21 (90). The usefulness of conducting a genome-wide search in the mouse model is its ability to control for environmental factors confounding all studies of asthma genetics in man a complete lack of control over genetic variation in man. Four of the five susceptibility loci identified in the Cookson study are supportive of the linkage studies in man, and future mouse studies may also prove useful in teasing out gene–environment interactions. Given the many susceptibility loci identified by linkage analysis, combined with the great strides achieved as part of the Human Genome Project to sequence the entire human genome, a great deal of effort has focused on the next stages of positional cloning: honing in on candidate genes by fine mapping, followed by screening the sequence (if known) of allelic variants in known genes, and analyzing the function of these variants in the context of the disease of interest. Much of the work focusing on allelic variants in candidate genes for allergic disease has been devoted to loci for which there was, a priori, preliminary evidence for linkage. As described above, in the earliest candidate gene studies, loci were selected by the mapping of Th2-type cytokines (e.g., the IL4 gene cluster in 5q31–q33) and receptors e.g., the high-affinity IgE receptor on 11q13), and “recognition” genes (e.g., the HLA-D complex on 6p21). More recently, researchers have considered genes encoding transcription factors (e.g., STAT6) and other mediators of inflammation (e.g., 5-lipoxygenase). Rosenwasser et al. (56) obtained evidence in families with asthma that a polymorphism in the IL4 promoter is associated with elevated total ser