ARTICLE

TOP ABSTRACT Article REFERENCES

Asthma is a disorder characterized by reversible airflow obstruction and bronchial hyperresponsiveness (BHR) to a variety of environmental stimuli (1). Although environmental factors are clearly important determinants of asthma susceptibility, a genetic component for asthma has been suggested by the results of both family and twin studies (2, 3). Using candidate gene approaches, linkages for asthma and asthma-associated phenotypes have been reported. In addition, the results of genome-wide screening have been reported for atopy (4) and more recently for asthma (5). It has now become clear that there are multiple regions of the genome that are likely to contain susceptibility genes for asthma and associated phenotypes that include BHR and atopic parameters (total serum immunoglobulin E [IgE] levels and allergy skin-test responses).

The major objective of the Collaborative Study on the Genetics of Asthma (CSGA), a multicenter study funded by the National Heart, Lung, and Blood Institute with the assistance of the National Institute of Allergy and Infectious Diseases, was to perform a genome-wide screen in families from different racial groups (caucasians, African-Americans, and hispanics). The initial genome screen of 360 markers spaced at approximately 10 cM intervals included 261 affected sibling pairs and their parents (140 families). This group represents approximately the first 40% of the total families that have been studied. All asthmatics met diagnostic criteria, which included a past history or physician's diagnosis of asthma, the presence of asthma symptoms, and evidence of BHR or reversibility of airflow obstruction. Currently, analysis has been performed for the asthma phenotype using affected sibling pairs. Additional data on associated phenotypes will be analyzed when the genome screen is completed in the remaining families.

Comparing the results from the CSGA genome screen and the one performed by Daniels and co-workers (4) on quantitative measures of allergy and asthma reveals evidence for linkage for several regions previously described for asthma, atopy, or associated phenotypes: chromosomes 5q, 6p, 11q, 12q, 13q, and 14q.

Chromosome 5q

The initial reports for linkage to chromosome 5q was found using a candidate gene approach; there are multiple genes on chromosome 5q that may be important in the regulation of IgE and the development or progression of inflammation associated with allergy and asthma. They include several pro- inflammatory cytokines (interleukins [IL] IL-3, IL-4, IL-5, IL-9, IL-13), the ₂-adrenergic receptor, the glucorticoid receptor lymphocyte (GRL), leukotriene C4 (LTC4) synthase, and several other candidates (6). Linkage to 5q has been observed in several populations for different 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 reported for regulation of total serum IgE levels (7).

In the Dutch population, significant evidence for linkage for total serum IgE levels was found for several markers using both sibling pair analysis and the lod score approach (8). Sixty percent of families are linked with a multipoint lod of 7.6 at 6 cM from the marker D5S1480 (9). Linkage of a susceptibility locus for BHR mapping to this same region of 5q in the Dutch families has been reported (10). When total serum IgE levels were included as a covariate, significant evidence for linkage for BHR was obtained, suggesting that there may be separate loci on 5q for these two important components of the asthma phenotype. Evidence for linkage of the asthma phenotype to this region has also been obtained in the same set of families (11).

A significant association between an IL-9 polymorphism and total IgE levels was observed in a sample of 131 randomly ascertained families (12). In the Dutch families, a novel polymorphism in exon 5 of IL-9, C to T nucleotide substitution at position 4130, that results in an amino acid change (Thr to Met) was found by direct DNA sequencing of probands (9). However, the frequency of this polymorphism was similar in probands (21.7% are CT, freq[T] = 10.9%) and in spouses (19.5% are CT, freq[T] = 9.8%), and there was no difference in mean total IgE levels between these affected and unaffected groups. Evidence for linkage and association to 5q and to specific alleles in the IL-4 and IL-9 genes have been recently described in a study of a general population (13). It will be necessary to study the different polymorphisms described in each of these studies as well as in different samples of families to determine whether linkage disequilibrium is present within the gene and whether these polymorphisms are found in other populations associated with the allergic or asthmatic phenotypes.

The ₂-adrenergic receptor maps to 5q, and two common polymorphisms in this gene may be associated with asthma severity and nocturnal asthma (14). It is difficult to detect significant associations because these are common polymorphisms (present in > 20% of the population) and these two structural polymorphisms are in linkage disequilibrium (14). In a study evaluating asthma in children, the Gln 27 polymorphism was found to be associated with an increased frequency of childhood asthma (15). No difference in the frequency of the Gln 27 polymorphism was found in a study of patients with fatal or near-fatal asthma (16). The previously reported association of the Gly 16 polymorphism with nocturnal and steroid-dependent asthma (14) was not found in these patients with fatal or near-fatal asthma (16). The function of these polymorphisms in influencing the expression of asthma phenotype or severity and potentially the responses to therapy will require further study.

Chromosome 6

The human leukocyte antigen (HLA) region and specific HLA haplotypes have been correlated with several measures of the allergic phenotype in a number of studies (3, 17, 18). There is a strong relationship between immune response to several highly purified allergens and specific DR and DQ haplotypes (19). In addition to the HLA locus, the gene for tumor necrosis factor alpha (TNF-) is located in this region and may represent a candidate gene for allergy and asthma. Linkage to chromosome 6 was reported for several markers and total eosinophil count in a genome screen of atopic families, and preliminary evidence for linkage to asthma was observed in Hutterite pedigrees (20). In the CSGA, there was evidence for increased allele sharing for markers on 6p21.3-23 (p = 0.01) in the caucasian sibling pairs with asthma. Because 75% of the family members with asthma had at least one positive skin test, this finding may be similar to previous observations of the association between atopy and the HLA loci, or may be related to TNF- gene function.

Chromosome 11q

Evidence for linkage of a broadly defined allergic phenotype to markers on chromosome 11q was first described in 1989 (21). In a later study, investigators postulated that sequence variants in the FcRI- gene might increase the risk for developing allergy and possibly even asthma (22). The postulated sequence variant (Leu181) was not found in several other studies (3, 23, 24). This inconsistency raises the issue of whether sequence variants in another gene on 11q confer susceptibility to allergic phenotypes. An association has been reported between specific alleles at two markers distant from the FcRI- and total IgE levels and BHR, suggesting that the FcRI- gene may not be the appropriate gene (12). Evidence for linkage was not found in the CSGA and Dutch families although such evidence was reported by Daniels and colleagues (4). These studies illustrate the difficulty in elucidating the gene responsible for an observed linkage, since sequence variants at one gene may only confer a moderate increase in risk in some individuals and may be difficult to detect in different population samples.

Chromosome 12q

Evidence for linkage to 12q of both asthma and total IgE levels was reported in an Afro-Caribbean population and in an Amish population for the regulation of total IgE levels (25). In the CSGA, evidence for linkage was observed for the asthma phenotype in two of the three racial groups studied: caucasians and hispanics. For both asthma and allergic phenotypes studied in a sample of random and multiplex asthma families (26), linkage has been reported for markers telomeric to those reported by Barnes and co-workers (25). There are several candidate loci in the region of chromosome 12q, including interferon gamma (IFN-), nitric oxide synthase (NOS1), a mast cell growth factor, and leukotriene A4 hydrolase (LTA4H), which is involved in leukotriene synthesis. As with the other chromosomal regions, evidence for linkage was reported over a wide region and fine mapping studies are required to determine whether one or more gene(s) account for this linkage.

Chromosome 13

The linkage to chromosome 13q may be one of the first linkage described for total serum IgE levels. In 1985, preliminary evidence for linkage to Esterase D and IgE levels was reported (27). Evidence for linkage of the atopic phenotypes has been reported by Daniels and co-workers (4). For the asthma phenotype, evidence for linkage (p = 0.001 multipoint analyses) was observed in the CSGA caucasian families (5). In the Hutterite population, there is evidence for linkage, and preliminary evidence for linkage disequilibrium with specific marker alleles has been described (28).

Chromosome 14q

In a sibling pair study for atopy, linkage to chromosome 14q was reported in the region of the T cell antigen receptor and NFB-1, an important immunoregulatory factor (29). Evidence for linkage to 14q was also obtained from the genome screen of the CSGA caucasian families (p = 0.006) (5), although further evidence for linkage was not reported by Daniels and colleagues (4). Fine mapping studies have not been completed in the CSGA families, so it is not yet known whether this linkage localizes to the same region as the initial linkage or to the region of NFB-1.

Novel Regions of Interest

Six novel regions were detected in the genome screen performed by the CSGA (5). In African-Americans, chromosomes 5p15 (p = 0.0008) and 17p11.1-q11.2 (p = 0.0015) showed evidence for linkage based on affected sibling pair analysis in 43 families. In 18 hispanic families, preliminary evidence for linkage was found for chromosomes 2q33 (p = 0.0005) and 21q21 (p = 0.004). In 79 caucasian families, 11p15 (p = 0.009) and 19q13 (p = 0.001) showed evidence for linkage. Evidence for two additional regions (chromosomes 7 and 16) was reported by Daniels and co-workers for different phenotypes including total IgE levels, BHR, and eosinophil count (4). The evidence for novel regions from both of these studies requires replication in additional families or populations before concluding that there are susceptibility genes in all of these chromosomal regions. Studies in multiple racial groups are needed to determine whether there are racial differences for genetic susceptibility to asthma and atopy. It is possible that the frequencies of a specific gene may differ across racial groups, accounting for these inconsistent linkage findings.

Many of the novel regions, and all of the previously described ones, contain relevant candidate genes for allergy and asthma. Although all of these regions probably do not contain susceptibility genes for asthma, it is clear that there are multiple genes present for both asthma and allergy. Some of these genes may affect the expression of the asthmatic or allergic phenotype or may interact with environmental exposures or pharmacologic interventions to modulate the natural history of these disorders. This has been observed with the ₂-adrenergic receptor and perhaps for genes that regulate leukotriene synthesis (14, 30). It is very likely that susceptibility to developing asthma is due to not only one of these genes in a given family (genetic heterogeneity), but to multiple genes that interact with each other and environmental factors to determine the expression of the asthmatic or atopic phenotype.

Once linkage is confirmed in complex genetic disorders such as asthma or clinical allergic disease, finding the actual gene is a complex process. Fine mapping studies will be required in regions where linkages have been replicated. These approaches will require further clinical characterization of asthma and relevant associated phenotypes. Molecular techniques will involve obtaining a physical map of the region of interest, and saturating the region with additional markers in order to localize genes within the area using complex linkage techniques. If the population group is isolated, linkage disequilibrium may be very helpful in narrowing the region. Physical mapping and detecting mutations in candidate genes, as well as evaluating mutations in novel genes, will be necessary. These positional cloning approaches will require sophisticated genetic analysis to narrow the region and perform association studies accurately. Even before isolation of a specific gene, biologic studies are necessary to evaluate the relevant candidate genes that fall within a confidence interval for the observed linkage. After identifying sequence variants, gene-gene and gene-environment studies will be necessary to understand the overall function of the gene in the expression of allergy and asthma.