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Human Molecular Genetics, 2003, Vol. 12, No. 20 2577-2585
DOI: 10.1093/hmg/ddg290
© 2003 Oxford University Press

LD mapping of maternally and non-maternally derived alleles and atopy in Fc{varepsilon}RI-ß

James A. Traherne{dagger}, Michael R. Hill{dagger}, Pirro Hysi, Mauro D'Amato, John Broxholme, Richard Mott, Miriam F. Moffatt and William O.C.M. Cookson*

The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7BN, UK

Received May 10, 2003; Accepted August 15, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Polymorphisms in the ß chain of the high affinity receptor for IgE (Fc{varepsilon}RI-ß, MS4A2) are consistently associated with traits underlying asthma and atopy (immunoglobulin E-mediated allergy). However, the causal variants and haplotypes underlying disease have not yet been identified. Maternal effects, with association confined to maternally derived alleles, have been shown in some studies but not in others. We have therefore extended the known sequence and systematically detected polymorphisms across an 18.1 Kb genomic region that includes Fc{varepsilon}RI-ß. Association testing in two panels of subjects showed the presence of single-nucleotide polymorphisms (SNPs) affecting prick skin tests and specific IgE responses in several clusters. Stepwise analyses indicated that the clusters represent independent effects. Interferon regulatory factor 2 (IRF-2) sites were altered by significantly associated SNPs in two regions. Strong association to maternally derived alleles was seen in one panel of subjects and not in the other. Maternal and non-maternally derived associations tended to share the same SNP clusters, but associations were stronger in the presence of maternal effects. Two regions of increased CpG concentration were identified in Fc{varepsilon}RI-ß. One of these approximated a SNP cluster that showed strong association and maternal effects, providing a potential substrate for epigenetic effects.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Atopy is a common familial syndrome that is due to interacting genetic and environmental factors. The atopic diseases include asthma, atopic dermatitis and allergic rhinitis and affect more than 10% of individuals in western populations (1). Atopy is characterized by increased total serum immunoglobulin E (IgE) concentrations and by elevations of IgE specific to common allergens such as house dust mite (HDM) proteins and grass pollen. Allergen-specific IgE may be detected by ELISA or RAST measurement of serum titres, and by prick skin tests (PST) in which minute amounts of allergen are introduced into the epidermis and the resulting skin wheal is quantified. The complex relationships between atopic diseases and these intermediate phenotypes can be dissected genetically in multivariate models (2,3), and quantitative phenotypes are effective surrogates for disease in linkage and association studies.

The high-affinity receptor for IgE (Fc{varepsilon}RI) links pathogen- or allergen-specific IgE with cellular and immunologic functions by activation and degranulation of mast cells and other cells (4). The consequent release of multiple mediators results in an acute inflammatory reaction, typified by the PST response in the skin or acute bronchospasm in the lung.

Linkage studies of atopic IgE responses and bronchial-responsiveness to markers on chromosome 11q12–13 have previously identified the gene encoding the beta chain of Fc{varepsilon}RI (Fc{varepsilon}RI-ß or MS4A2: OMIM 147138) as modifying the prevalence of atopic disease (5–9). Preferential transmission of atopic diseases from mothers rather than fathers is well recognized (reviewed in 10), and in several studies linkage has only been observed to alleles derived from the mother (5–8,11).

The Fc{varepsilon}RI-ß protein acts as an amplifying element of the high-affinity IgE receptor response to activation (12) and in addition stabilizes the expression of the receptor on the mast cell surface (4). Polymorphism in Fc{varepsilon}RI-ß is therefore in an ideal position to modify Fc{varepsilon}RI signalling in response to allergens.

Fc{varepsilon}RI-ß is composed of seven exons and six introns, spanning ~11 Kb (13). Initial sequencing of the coding regions of Fc{varepsilon}RI-ß detected polymorphisms within exon 6 (L181I and L183V), which strongly associated with atopic asthma and measures of atopy in British and Australian subjects (14). Although these variants have been reported in Kuwaiti Arabs (15), South African Blacks (16) and Italians (17), they have been difficult to identify in other population samples and their status is uncertain. Another coding polymorphism in Fc{varepsilon}RI-ß located in exon 7, E237G, associates with elevated bronchial reactivity and skin test response to common allergens (18), rhinitis and IgE levels (19), asthma (20), and atopy and bronchial hyper-responsiveness (21). These coding polymorphisms do not alter receptor function (22,23).

A limited number of non-coding polymorphisms in Fc{varepsilon}RI-ß have been identified, and some of these show associations with asthma (20,24), histamine release from mast-cells (25), bronchial hyper-responsiveness (26), atopic dermatitis (27) and elevations of the total serum IgE concentration in Caucasian (28) and Aboriginal Australians (28).

The phenotypes with which Fc{varepsilon}RI-ß polymorphisms have shown association are therefore varied. Simple changes in the sensitivity of mast cells to degranulation might be expected to influence prick skin tests and the presence and severity of allergen-induced asthma. However, skin test and airway responses to allergen will also depend on the presence and level of IgE specific to the allergen (29). In this study, as a proxy for mast cell sensitivity, we have therefore examined associations to allergen-induced prick skin tests (PSTI) with the specific IgE serum titres (RASTI) included as a covariate.

The observed associations of Fc{varepsilon}RI-ß SNPs to elevated total and specific serum IgE suggest as yet unrecognized mechanisms influencing IgE synthesis, that lie beyond simple allergen-induced degranulation. We have used the RASTI to quantify these effects on specific IgE synthesis.

Several studies have shown association to be limited to maternally derived alleles (14,17,27,30–32), consistent with the previously observed maternal linkage to the locus. However, in many other studies either positive linkage or association to chromosome 11q13 and Fc{varepsilon}RI-ß did not show a maternal bias. This suggests that a variable process underlies the maternal effect at this locus.

Thus, despite convincing evidence for the importance of the Fc{varepsilon}RI-ß locus to atopic disease, the polymorphisms which modify its function remain to be identified, and no mechanism has been found to explain the maternal effects.

We therefore extended the previous published sequence of Fc{varepsilon}RI-ß (13) and have carried out an extensive search for sequence variants. The newly identified polymorphisms have been assessed for association to measures of atopy in two panels of families: a panel recruited from the general population, and a panel of families recruited through a proband with asthma. The first panel of subjects does not demonstrate maternal effects, whereas the second shows strong maternal effects at Fc{varepsilon}RI-ß and other loci (8).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Polymorphism within Fc{varepsilon}RI-ß (MS4A2) and surrounding sequence
A physical map of the locus was constructed and sequenced, to form a contiguous 18 098 bp region. A total of 38 sequence variants were detected in the sequence (see Supplementary Material). The nature of the sequence change and its position relative to the first nucleotide of the Fc{varepsilon}RI-ß cDNA transcript, labelled as base +1, are given in Figure 1 and Table 1. The base immediately before +1 was labelled as -1. Thirty-five of the sequence variants identified involved a single base substitution, two were simple dinucleotide (CA) repeats and one, {varepsilon}ß-1862{Delta}TGC, involved a three base deletion. The {varepsilon}ß+6858 polymorphism (E237G) (18) confers the only coding change found.



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  		Figure 1. Fc{varepsilon}RI-ß/MS4A2 genomic structure and sequence polymorphism. Polymorphisms genotyped in the study panels are labelled in blue. Base A of the ATG initiator Met codon of Fc{varepsilon}RI-ß is denoted nucleotide +1. Dinucleotide repeats are in brackets. Promoter Scan was used to predict forward (F1 and F2) and reverse (R) strand promoter sequences.

 

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  		Table 1. Polymorphisms within Fc{varepsilon}RI-ß/MS4A2 and the surrounding sequence
 
Six of the sequence variants have previously been reported in the literature, {varepsilon}ß-211 (originally named -109) (33), {varepsilon}ß+1343 (RsaI-ex2) (14,27), the dinucleotide repeat {varepsilon}ß+5026 (FCERIB) (5,8), {varepsilon}ß+3934 (34), {varepsilon}ß+6858 (E237G) (18) and {varepsilon}ß+9424 (RsaI-ex7) (27). dbSNP accession numbers had previously been assigned to 18 of the SNPs detected (Table 1). We did not find the previously reported L181I and L183V polymorphisms (14).

Thirty-one single-nucleotide polymorphisms (SNPs) and one dinucleotide repeat were typed in the two panels of families. The remaining SNPs were not amenable to genotyping by PCR and restriction digestion. The {varepsilon}ß+3264 dinucleotide repeat was of low heterozygosity and was also not typed (Table 1).

Linkage disequilibrium within Fc{varepsilon}RI-ß (MS4A2)
The regional distribution of linkage disequilibrium (LD) within Fc{varepsilon}RI-ß was seen to be irregular, but approximately distributed into proximal and distal islands (Fig. 2). Three SNPs in the promoters of the gene ({varepsilon}ß-3383, {varepsilon}ß-2384 and {varepsilon}ß-2381) showed strong LD with each other but little LD with the rest of the gene. The adjacent {varepsilon}ß-1964{Delta} deletion, however, showed some LD with almost all markers studied, perhaps indicating a relatively recent origin. Other SNPs from position -756 to +13948 tended to be in strong to moderate LD with each other, although {varepsilon}ß+8033 (minor allele frequency 0.47) and {varepsilon}ß+12209 (minor allele frequency 0.05) showed low levels of LD with most markers.



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  		Figure 2. Linkage disequilibrium within Fc{varepsilon}RI-ß/MS4A2. Pairwise estimations of D' are shown from unrelated subjects (the parents), on a scale of 1 (complete LD: red) to 0 (blue). Marker positions are shown as a schematic rather than at actual distances apart. The lower insert illustrates the distribution of LD with true distances.

 
Associations with atopy phenotypes
Sixty-two per cent of the subjects in panel 1 were atopic and 18% were asthmatic, compared with 67% atopic and 45% asthmatic in panel 2. The mean loge IgE concentration was 3.87; variance ({sigma}2)=2.76 in panel 1 and 4.18; {sigma}2=3.23 in panel 2. This is consistent with the selection of panel 1 from the general population, and panel 2 through out-patient clinics. However, the mean PSTI and RASTI were similar in the two panels (mean PSTI 2.3; {sigma}2=9.1 in panel 1 and 2.7; {sigma}2=12.7 in panel 2; and mean RASTI 2.1; {sigma}2=6.8 in panel 1 and 1.8; {sigma}2=4.8 in panel 2).

Markers were first tested for single-locus association by variance components with QTDT in all subjects (parents and children). We have examined associations to the PSTI with RASTI included as a covariate (Table 2) as a proxy for mast cell degranulation.


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  		Table 2. Positive results of allelic association tests in two panels of subjects. The results of association testing to all alleles are compared with the results of testing to maternally derived alleles. Panel 1 was derived from a general population sample and panel 2 was recruited through a proband with atopic asthma
 
Four clusters of SNPs were associated to the PSTI/RASTI in both panels (Table 2). These corresponded approximately to the leader sequences and intron II; introns III, V and VI; the distal 3'-UTR and contiguous sequences; and SNPs in the distal end of the region. In general, the associations were stronger in panel 1. An association was observed in position -854 in the panel 1 subjects but not in panel 2, and weak associations were observed within the predicted F2 promoter only within the panel 2 subjects.

A stepwise procedure was then performed to determine whether SNPs in these regions independently contributed to the phenotype. The {varepsilon}ß+3934 SNP (P=0.006) was first included as a covariate in the QTDT analysis: the most significantly associated remaining SNP, {varepsilon}ß+9424 (P=0.004), was then included, then {varepsilon}ß+5565 (P=0.06), then {varepsilon}ß+1343 (P=0.07), at which step no significant associations remained. These results suggested that there are at least two independent effects within the locus, and possibly four.

These SNPs are all in partial LD with each other (Fig. 2). We therefore generated an extended haplotype of all four SNPs. {varepsilon}ß+1343*2/{varepsilon}ß+3934*1/{varepsilon}ß+5565*1/{varepsilon}ß+9424*1 (*2*1*1*1) was the most common haplotype, and was negatively associated with the PTSI/RASTI phenotype (Table 3). The *1*2*2*2 haplotype was the second most common, and was positively associated with PSTI/RASTI. Three other rarer haplotypes (*2*2*2*2, 1*1*1*1 and *2*2*1*1) were also positively associated with the phenotype. The {varepsilon}ß+3934*2 SNP (or closely neighbouring markers) is therefore present on most positively associated haplotypes; however the weakly positive association with *1*1*1*1 and the disproportionate strength of the association to the rare haplotype *2*2*1*1 provide further evidence that the {varepsilon}ß+3934*2 cluster is not the only determinant of positive associations with the locus.


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  		Table 3. Association of PSTI to {varepsilon}ß+1343/{varepsilon}ß+3934/{varepsilon}ß+5565/{varepsilon}ß+9424 haplotypes. Panel 1 subjects: RASTI included as a covariate
 
Maternal effects
Single locus transmission tests of association were then carried out with QTDT. In both panels of families weak associations were seen to the PSTI with RASTI as covariate (results not shown). This is consistent with reduced numbers of subjects (because transmission tests do not measure the relationship between phenotype and genotype in parents).

A test for the presence of maternal effects for the RASTI was positive in the panel 2 subjects (P<0.01) but not in panel 1. Association to maternally derived alleles was then examined (Table 2). Strong associations were seen to the RASTI and PSTI independently, but not in combination (Table 2 shows the results of testing the association to RASTI: the PSTI gave similar results).

These findings suggest that maternal effects mediated through this locus are modifying the IgE titres to specific allergens. This is in contrast to the PSTI/RASTI proxy measure of sensitivity of allergen-induced mast cell degranulation, which does not seem to show maternal modification.

The location of association with maternal effects was similar to that seen with non-maternal effects in both panels, and was concentrated into four regions. Step-wise analyses again indicated an independent effect of several regions (most significant marker step 1, {varepsilon}ß+3934, P=0.00004; step 2, {varepsilon}ß+5734, P=0.0002; step 3, {varepsilon}ß+1343, P=0.0023; step 4, {varepsilon}ß-211, P=0.006).

Potential sites of methylation
Epigenetic methylation of CpG residues is one potential mechanism for maternal effects. We therefore looked for excess concentrations of CpG segments in the sequence, using a permutation test to determine statistical significance. Two significant regions were detected, the first beginning at -1619 and ending at -1759 [Mott Island Score (MIS)=201; P=0.0079], and the second beginning at +10278 and ending at +10514 (MIS=260; P=0.0003; Fig. 1).

The first CpG concentration (MI1) contained {varepsilon}ß-1964{Delta}, which did not show convincing associations with either phenotype. However, the second concentration (MI2) approximated the highly maternally associated {varepsilon}ß+10062 SNP (Table 2).

Potential transcription factor binding sites
F1 and F2 promoters were identified by PROMOTERSCAN at -3462 to -3212, and -2554 to -2304 respectively (Fig. 1). A reverse promoter (R) was identified between +9637 and +9387. The sequence was examined for potential transcription factor (TF) binding sites that were altered by the presence of SNPs, using the MatInspector program (35). Interferon regulatory factor 2 (IRF-2) sites were identified in two regions with significantly associated SNPs, and a number of OCT-1 sequences were observed in the F1 and F2 promoters (Fig. 3).



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  		Figure 3. Potential TF binding sites affected by SNPs. TFs corresponding to atopy-associated SNPs are shown with background shading. SNPs are shown in bold in the consensus recognition sequence. Bases shown in red appear in a position exhibiting high conservation. Bases shown in capital letters denote the most highly conserved positions of the consensus recognition sequence.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Our study is likely to have identified all common polymorphism in the genomic sequence of the Fc{varepsilon}RI-ß gene and upstream and downstream DNA. The sequencing of 12 unrelated individuals has given us 99% probability of identifying SNPs with a minor allele frequency >=0.1 and 95% probability of identifying SNPs with a minor allele frequency >=0.05 (36) and no additional SNPs are to be found in public databases.

The results of the analyses show the presence of SNPs affecting prick skin tests and specific IgE responses in several clusters. Our stepwise analyses indicate that the clusters represent independent effects. Further experiments will be required to define which individual SNPs are disease-causing. The examination of haplotypes of the most strongly associated SNPs identifies a single common protective haplotype, and one common and several rare susceptibility haplotypes. The haplotypes also do not suggest that a single SNP is responsible for association of atopy phenotypes to this locus. Further dissection of the effects of these haplotypes will require analyses in larger population samples.

Although most of the SNPs in the body of the gene were in partial LD with each other, the LD was incomplete and irregularly distributed and declined with distance. The findings are similar to our studies of dense SNP maps at other loci (37,38). These results are not consistent with the haplotype block hypothesis (39,40), and the typing of a limited number of SNPs from this gene might lead to misleading results from association testing.

We have only identified one coding polymorphism, {varepsilon}ß+6858 (E237G) and this does not have functional effects on Fc{varepsilon}RI receptor expression or signalling (22). The Fc{varepsilon}RI-ß chain is not essential for Fc{varepsilon}RI function and does not possess autonomous cell activation capacity, but it augments the surface expression of the receptor (22) and acts as a 12–30-fold amplifier of Fc{varepsilon}RI-{gamma} mediated cell activation signals (12,22,41). Receptor function may therefore be modified by variation in the level of ß chain expression, or in the level of the recently recognized truncated form, ßT, which regulates receptor surface expression (42).

The study has identified several potential TF binding sites that are affected by SNPs. In silico prediction of TF binding sites is non-conservative, and any predictions by MatInspector and similar programs should be the prelude to functional studies (43). Nevertheless, it may be of interest that interferon regulatory factor 2 (IRF-2) sites are identified in two regions with significantly associated SNPs. IRF-2 is a member of a family of transcriptional factors involved in the modulation of cellular responses to interferons and viral infection as well as in the regulation of cell growth and transformation. IRF-2 polymorphisms have been associated with atopic eczema (44) and IRF-2 knockout mice show defects in CD8+ T cells and spontaneous development of an inflammatory skin disease (45). We also found polymorphisms in the OCT1 sites of the F1 and F2 promoters, but these SNPs showed weak associations with PSTI or RASTI.

We observed the presence of maternal effects in one population of subjects but not the other. The two panels had a similar prevalence of atopy, measured by PSTI and RASTI, but total serum IgE levels were higher and there were many more asthmatics in the panel that showed maternal effects (panel 2). The panels were of similar sizes, so the maternal effects in panel 2 were not attributable to differences in power. Selection for parental phenotype might have biased allele frequencies and subsequent transmission ratios, but the panel 2 families were identified through second-generation probands and not through the parents. The information content of maternal and paternal alleles was similar, and similar results were seen if the results are categorized and the allele transmissions counted by other programs (data not shown). The analyses of association indicated that the maternal effects are operating through the same SNPs as those which lead to non-maternal associations. However, in the presence of maternal effects associations were stronger and appeared to influence a different phenotype (elevation of specific IgE levels in the serum as opposed to mast cell sensitivity to allergen). This might reflect the involvement of Fc{varepsilon}RI signalling in the induction or maintenance of IgE responses to specific allergens.

Parent-of-origin effects have been observed at other loci influencing allergic disease (8,46,47) and in other immunological disorders such as type I diabetes (48), Crohn's disease (49), rheumatoid arthritis (50) and IgA deficiency (51). The strength of parent-of-origin effects in type I diabetes had also been observed to differ among family collections from different populations (52). No mechanism has been identified for these phenomena. Interaction between maternal and fetal immune systems, and genomic imprinting of disease genes, are two possibilities (53). The two regions of increased CpG concentration that we have identified in Fc{varepsilon}RI-ß provide a potential substrate for epigenetic effects.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Physical mapping
Human genomic PAC clones containing Fc{varepsilon}RI-ß/MS4A2 sequence were identified through hybridization-based library screening using a 690 bp cDNA clone, covering exons 2–7 of Fc{varepsilon}RI-ß, and a 662 bp genomic PCR product covering the 5' untranslated region and exon 1. Positive clones were mapped by FISH to confirm their chromosomal location. A restriction map was generated from the isolated PAC clones to derive a consensus contig. The position of Fc{varepsilon}RI-ß was located by hybridizing the 690 bp cDNA probe onto the restriction fragments of the clones.

A vectorette PCR technique adapted from Munroe et al. (54) was used to extend the sequence of Kuster et al. (13) and isolate (CA)n repeats. DNA sequencing used fluorescently labelled dye-terminator chemistry and an Applied Biosystems 377 sequencer. This provided an additional 3213 bases of sequence upstream and 3580 bases of sequence downstream of the previously described sequence (13). All sequences were aligned (GAP4 program, STADEN package) to produce 18098 bases of continuous sequence for polymorphism detection (available as Supplementary Material).

Polymorphism detection
Variants within Fc{varepsilon}RI-ß and the surrounding region were detected by sequencing 12 unrelated British Caucasian individuals. The sequences were analysed for variants using PhredPhrap (www.phrap.org). Additional SNPs within Fc{varepsilon}RI-ß were located on dbSNP (www.ncbi.nlm.nih.gov/SNP/) and their presence checked in a different set of unrelated individuals by restriction fragment length polymorphism (RFLP) and sequence analysis.

Subjects
Association analyses were carried out using two panels of subjects. Panel 1 subjects consisted of 1004 Caucasian individuals in 230 nuclear families randomly selected from a population sample in the rural West Australian town of Busselton (30). Panel 2 contained 380 subjects in 67 nuclear and seven extended pedigrees from the UK, recruited through probands attending hospital clinics with symptoms of asthma or atopic disease (55).

Phenotyping
The same protocols were used to test both panels of families. Prick skin tests to HDM and mixed grass pollen (less the response of negative controls), specific IgE titres to HDM and Timothy grass and the total serum IgE were measured as previously described (30). A PSTI was calculated as the sum of the prick skin test results to HDM and grass mix (95% of individuals who were atopic reacted either to HDM, or to grass pollen or both). The RASTI was defined as the sum of the RAST scores for the serum IgE concentration specific to the same two allergens.

Genotyping
SNPs were genotyped by RFLP analysis of PCR products. The {varepsilon}ß-1964{Delta}TGC deletion was genotyped by ARMS analysis. Detailed PCR and RFLP conditions are given in Table 1 in Supplementary Material. Two individuals checked genotypes independently without knowledge of phenotype. The study panels were not genotyped for SNPs {varepsilon}ß-3226, {varepsilon}ß-528, {varepsilon}ß+1798, {varepsilon}ß+11340, {varepsilon}ß+12400 either because the polymorphisms were rare or because they could not be typed by restriction digestion. The {varepsilon}ß-3264 dinucleotide repeat also was not typed in the full sample because of its low information content. The dinucleotide repeat {varepsilon}ß+5026 (FCERIB) was genotyped as described previously (8,55).

Statistical analysis
Tests of association with the quantitative traits were performed in a variance components framework using the QTDT program (56). Associations were first tested using information from all family members (‘total association’) and then transmission tests of association were carried out. The presence of a parent origin effect was first established by a modified Weinberg likelihood test (57) in which a model including ordered mating types and association including dominance was compared with a model that allowed heterozygotes to be different depending on which parent was being tested (47). Maternal effects were then examined by QTDT (56). It was recognized that the transmission tests were less powerful than the total association model because they only used information from children. The dinucleotide repeat {varepsilon}ß+5026 was tested for association using the multi-allelic option of QTDT.

In order to further refine the localization of the primary disease-associated alleles, a stepwise procedure was performed in which the most significantly associated SNP was included as a covariate in the QTDT analysis. The analysis was then repeated, and the next most significantly associated remaining SNP was included as an additional covariate, until no significant associations remained.

Haplotypes were generated by the MERLIN program (58). Pair-wise D' measurements were made between SNPs from the parental (i.e. unrelated subject) haplotypes and LD across the locus was plotted by the GOLD program (59). For each marker pair, GOLD plotted the colour-coded pairwise disequilibrium statistics at their Cartesian co-ordinates, and the plots were completed by interpolation.

CpG islands were detected by searching for high-scoring segments using an ungapped Smith–Waterman algorithm (60,61) in which each CpG dinucleotide scores +20 and all other dinucleotides score -1 (PERL source code available on request). A permutation test was used to determine statistically significant CpG islands: in order to model the dependence between nearby nucleotides, the sequence was divided into non-overlapping 10 bp segments, the order of which was permuted. The highest-scoring CpG island in the permuted sequence was recorded, and process was repeated 1000 times until the empirical distribution of CpG island scores could be estimated with precision.

Potential binding sites for transcription factors were identified on forward and backward strands of genomic DNA using the MatInspector program (35) (www.genomatix.de). The program produces an RE (random expectation) value for each individual consensus binding site matrix, based on an expectation for the number of matches per 1000 bp of random DNA sequence. The matrix similarity is calculated as described (35). A perfect match to the matrix gets a score of 1.00 and a ‘good’ match to the matrix usually has a similarity of >0.80. We considered sequences with an RE value <0.05 and a matrix similarity score >0.80 to be of interest.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
The study was funded by the National Asthma Campaign and the Wellcome Trust. We are grateful to the people of Busselton and the Busselton Foundation for allowing us to study the panel 1 subjects. Elaine Levy carried out the Fish analyses of BAC and PAC clones.


   FOOTNOTES
 
* To whom correspondence should be addressed. Fax: +44 1865287578; Email: wocc{at}well.ox.ac.uk Back

{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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