Linkage and Candidate Gene Studies in Asthma

MIRIAM F. MOFFATT and WILLIAM O. C. M. COOKSON

Asthma Genetics Group, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom

	INTRODUCTION

TOP INTRODUCTION REFERENCES

Study of the genetics of asthma will increase understanding of the etiology and pathophysiology of the disease. The early identification of children at genetic risk of asthma will open new approaches to the prevention of disease. The involvement of particular genes may identify a particular clinical course and response to therapy. Eventually, genetics will lead to new pharmacological treatments for asthma.

We have used two approaches to identify genes that influence asthma. The first of these is "positional cloning," which relies on the localization of disease genes to particular chromosomal segments by genetic linkage, followed by strategies to isolate the disease gene from the identified region. The second approach is testing variants of candidate genes for associations in affected and unaffected individuals. Our experience with various loci is described below.

	CHAIN OF THE HIGH-AFFINITY RECEPTOR FOR IMMUNOGLOBULIN E

Linkage of atopy, defined by IgE responses, to the chromosome l1q13 marker D11S97 was first found in 1989 (1) and has been widely replicated (2).

Affected sibling-pair analysis showed that linkage of atopy to chromosome 11 markers was exclusively maternal in our subjects (6, 7). This is likely to correspond to the maternal effect seen in clinical studies (8). The recognition of the maternal effect allowed mapping of the atopy gene centromeric to the original D11S97 marker and the demonstration that 60% or less of families with symptomatic atopy can be influenced by the chromosome 11 atopy gene (7, 14). It was also followed by the localization of the  chain of the high-affinity receptor for IgE (FcRI-) to the same region, in close linkage to atopy (14).

The first reported FcRI- polymophisms were known as Leu 181/Leu 183 and Leu 181 (15), now designated I181L/ I183V and I181L. Maternal inheritance of both these variants was associated with severe atopy. However, there has been great difficulty in detecting these variants, the structural reasons for which we are currently investigating.

A further search through the coding regions of FcRI- has identified a coding polymorphism in exon 7 (16). An adenine to guanine substitution changes amino acid residue 237 from glutamic acid to glycine (E237G) in the cytoplasmic tail of the protein. E237G is predicted to introduce a hydrophobicity change within the C-terminus of FcRI-. It is adjacent to the immunoreceptor tyrosine activation motif (ITAM) and may affect the intracellular signaling capacity of FcRI. The variant has been identified in diverse populations and is easily assayed. E237G was detected in 53 subjects (5.3%) from an Australian general population sample of 1004 individuals (16). E237G-positive subjects had elevated skin-test responses to grass (p = 0.0004) and house dust mite (p = 0.04), RAST to grass (p = 0.002), and bronchial reactivity to methacholine (p = 0.0009). The relative risk of individuals with E237G having asthma compared to subjects without the variant was 2.3 (95%CI 1.26-4.19; p = 0.005). E237G did not show a parent-of-origin effect in this population.

Although E237G associates with atopy and may be of functional importance, it cannot on its own or in combination with I181L/I183V explain the strength of the chromosome 11q13 linkage. Further functional polymorphism in and around FcRI- therefore remains to be discovered.

	T CELL RECEPTOR- AND HUMAN LEUKOCYTE ANTIGEN-DR

The development of disease in atopic individuals depends on the type of allergens to which they react (17, 18), so that children with house dust mite (HDM) allergy have a greater risk of asthma than children who respond purely to grass pollen. It is therefore of clinical interest to investigate the genetic control of the specific immunoglobulin E (IgE) response. Inhaled allergen sources such as HDM or grass pollens are complex mixtures of proteins. In the case of HDM, the two most important allergens are Der p I (25.4 kD) and Der p II (14.1 kD), each of which seems to have four major B cell epitopes (19, 20). Peptide mapping of Der p II has shown that T cell clones from different individuals may also react to common T cell epitopes (21).

The human leukocyte antigen and T cell receptor (TcR) genes are candidates for germ-line influences on specific allergen responses. The association of HLA haplotypes and ragweed allergy was the first human Ir (immune response) gene to be recognized (22), and HLA-DR restriction of IgE reactions to allergen is well documented (23, 24). However, the HLA genes on their own do not account for the differences in an individual's IgE reactions to allergen (24). Polymorphisms in the TcR genes influence the peripheral TcR repertoire (25- 27) and may affect the immune response to antigen. We have therefore examined the TcR loci for genomic restriction of IgE responses.

We have previously established linkage of specific IgE responses to the TcR-/ locus on chromosome 14 (but not to TcR-) in two sets of subjects (28). The TcR-/ region is complex (29) and contains many elements that might influence specific antigen recognition. Localization of these elements depends on associations between specific alleles and IgE responses. It has been previously demonstrated that V8 may be in excess in T cell clones reacting to HDM (30), and so we have now investigated a bi-allelic polymorphism in V8.1 (31) for association with IgE titers to HDM and its major antigens. In order to investigate possible interactions between HLA and TCR loci, the subjects were also HLA-DR typed.

In a panel of 400 subjects, allele 2 of the V8.1 polymorphism (V8.1*2) showed a significant association with higher IgE titers in Der p II (p = 0.006); a weak association was seen with Der p I (p = 0.057) (32). The association with V8.1*2 was confirmed in a second set of 400 subjects from the same population (p = 0.03) and was highly significant in the pooled subjects (p = 0.000). The IgE titers were approximately 25% higher in subjects with V8.1*2, in both groups and in the combined data.

In order to account for possible interacting effects with HLA-DRB1, multiple regression analysis was carried out with IgE titer to Der p II as the dependent variable, and V8.1 and the six most common HLA-DR types as independent variables. The results showed that both V8.1*2 and HLA-DRB1*1501 were positively associated with IgE titers in Der p II in both sets of subjects and in the pooled data (p = 0.0001 for both V8.1 and HLA-DRB1*1501 in pooled data). In the pooled data, the mean Der p II IgE titer when V8.1*2 and HLA-DRB1*1501 were together in the same subject was 1.14 ± 0.14 RAST classes, compared to 0.56 ± 0.025 when neither allele was present. This level of enhanced IgE response is likely to be of clinical significance.

These results indicate that germ-line elements in the TCR-V region interact with particular HLA-DR types to modify the response to foreign antigen.

	TUMOR NECROSIS FACTOR

Airway inflammation is a prominent feature of asthma (33, 34). The pro-inflammatory cytokine tumor necrosis factor (TNF) shows constitutional variation in the level of secretion, which is linked to polymorphisms within the TNF gene complex and the surrounding major histocompatibility complex (MHC) (35). Tumor necrosis factor is prominent in asthmatic airways (38). We have studied 413 subjects in 88 nuclear families from a general population sample for association of asthma and TNF polymorphisms (39). Ninety-two subjects were asthmatic, as defined by questionnaire. Asthma was significantly more common in subjects possessing allele 1 of the LT NcoI polymorphism (LT NcoI*1) (p = 0.005) and allele 2 of the TNF-308 polymorphism (TNF-308*2) (p = 0.004). The association was confined to the LT NcoI*1/TNF-308*2 haplotype, so that it was not possible to differentiate between the effects of LT NcoI and TNF-308 alleles. The HLA-DR locus was excluded as a cause of this association.

The ln IgE did not show association with LT NcoI or TNF-308 genotypes by analysis of variance, indicating that the association of the TNF polymorphisms with asthma is independent of atopy.

The results show that genotypes known to correlate with increased TNF secretion are associated with an increased risk of asthma, and they suggest that genetic influences on inflammation are part of the pathogenesis of the disease.

	GENOME-WIDE SEARCH FOR ASTHMA-ASSOCIATED TRAITS

In order to identify systematically genetic loci influencing asthma, our group has recently completed a genome-wide search for linkage to asthma-associated traits (40). The search used four quantitative parameters: the total serum IgE, the Skin-Test Index (STI), the peripheral blood eosinophil count, and bronchial responsiveness to methacholine (slope). A RAST index was also calculated, but added no information to the STI. To account for heterogeneity of phenotype (pleiotropy), the categorical trait of "atopy" was used, based on a combination of the STI, RAST index, and the total serum IgE.

Linkage to quantitative traits was tested by the Haseman-Elston sibling-pair technique, and to atopy, by affected sibling-pair methods.

Because the prevalence of atopy in Western populations is between 40 and 50%, we have found that the recruitment of families with asthma in multiple members results in samples in which 70% or more subjects are atopic, with severe loss of power to detect linkage (6). For this reason, the 80 families in the genome screen were selected from a population sample to include nonatopic members. It was also reasoned that detection of linkage to quantitative traits would be enhanced by inclusion of subjects in normal and abnormal ranges of the trait distributions. The 80 families contained a total of 203 offspring forming 172 sibling pairs. The families were initially screened with 269 markers (253 autosomal and 16 X-linked).

Six regions of potential linkage (p < 0.001) to autosomal markers were detected with one or more phenotypes on chromosomes 4, 6, 7, 11, 13, and 16 respectively. A Monte Carlo procedure was applied to calculate the number of false positive linkages. The expected number of regions of false positive linkage was 1.6 for p < 0.001, compared to six observed linkages, so that the observed number of regions meeting these statistical criteria for linkage was much larger than would be expected by chance.

Chromosomes 11 and 16 exhibited potential linkage to IgE levels. The chromosome 11 marker FCERB (40), a microsatellite in FcRI-, also showed linkage to the STI. The regions on chromosome 4 and 7 were linked to bronchial responsiveness, whereas the region on chromosome 6 (near the class I genes of the MHC) was linked to eosinophil counts. Weaker evidence of linkage (p < 0.01) was found with other phenotypes for the markers on chromosome 6 and 7. Markers from chromosome 13 around D13S153 showed evidence of linkage to the atopy phenotype in affected sibling-pair analyses.

The markers showing p < 0.001 for linkage were tested for replication in an additional panel of 77 nuclear and extended families recruited from clinics in the United Kingdom who had been previously used to map atopy on chromosome 11q13. They contained 215 offspring (268 sibling pairs) of which 61% were atopic and 56% asthmatic, reflecting their selection through clinics.

Linkage of asthma to FCERB (p = 0.003) and to chromosome 16 (p = 0.03) was seen in these families. Linkage with atopy was found to chromosome 13 (p = 0.003). Chromosomes 4, 11, and 16 showed significant differences in linkage between maternal and paternal alleles. Linkage to maternal meioses was seen between chromosome 4 and atopy (p < 0.05) and the total serum IgE (p < 0.001), and between chromosome 16 and atopy (p < 0.01) and asthma (p < 0.001). As already observed, FCERB showed strong maternal linkage to atopy (6), but a strong linkage to asthma was also seen (p < 0.00001), which has not been previously noted. The finding of maternal effects at several loci favors immunological interactions between mother and child rather than genetic imprinting or anticipation as a cause of these phenomena.

Thus, Monte Carlo simulations and the replication of positive results in a second set of subjects suggest that regions of true linkage have been identified by our study. The identification of the genes from all these loci, and those identified by other groups, remains a formidable task that will take several years of intense effort. Nevertheless, progress is being made.

	Footnotes

Correspondence and requests for reprints should be addressed to Dr. William Cookson, Asthma Genetics Group, University of Oxford, Nuffield Department of Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK.

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