WO2005017505A2 - Diagnosis of atopic disorder - Google Patents

Diagnosis of atopic disorder Download PDF

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Publication number
WO2005017505A2
WO2005017505A2 PCT/GB2004/003518 GB2004003518W WO2005017505A2 WO 2005017505 A2 WO2005017505 A2 WO 2005017505A2 GB 2004003518 W GB2004003518 W GB 2004003518W WO 2005017505 A2 WO2005017505 A2 WO 2005017505A2
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gene
polymorphism
polymorphisms
atopic
fcεri
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PCT/GB2004/003518
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French (fr)
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WO2005017505A3 (en
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William Osmond Charles Cookson
Miriam Fleur Moffat
Michael Richard Hill
James Arnold Traherne
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Isis Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • 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 characterised 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.
  • IgE Immunoglobulin E
  • 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.
  • PST prick skin tests
  • 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 ⁇ RI
  • the Fc ⁇ RI- ⁇ protein acts as an amplifying element of the high-affinity ⁇ g ⁇ . receptor response to activation (12) and in addition stabilises the expression of the receptor on the mast cell surface (4). Polymorphism in Fc ⁇ RI- ⁇ is therefore in an ideal position to modify Fc ⁇ RI signalling in response to allergens.
  • Fc ⁇ RI- ⁇ is composed of seven exons and six introns, spanning approximately 11 Kb (13).
  • Initial sequencing of the coding regions of Fc ⁇ RI- ⁇ detected polymorphisms within exon 6 (L181I and L183V), which is 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),
  • WO02/062946 discloses human and mouse proteins that span the cell membrane at least four times and share high levels of sequence identity with Fc ⁇ RI - ⁇ , CD20 and HTm4. These proteins are stated to reveal a new gene family, designated as MS4A.
  • Fc ⁇ RI- ⁇ gene designated as MS4A2
  • MS4A2 is disclosed as being useful in the treatment of atopic disorders, this is in the context of evaluating drugs that modulate the MS4A2 protein function.
  • WO97/08338 discloses the E237G polymorphism in exon 7 of Fc ⁇ RI- ⁇ . A method for diagnosing atopy based upon the identification of this polymorphism is also disclosed.
  • WO95/05481 discloses the identification of specific polymorphisms at amino acids 181 and 183, in exon 6 of Fc ⁇ RI - ⁇ . Van Hage-Hamsten et al, Clin. Exp. Allergy, 2002; 32: 838-842 and Kim et al, Clin. Exp.
  • a method for detecting whether a subject has or is predisposed to an atopic disease comprises: determining the presence in the subject of any of the genetic polymorphisms disclosed in Table 1.
  • an isolated polynucleotide useful for diagnosing whether a subject has or is predisposed to an atopic disease comprises at least 15 contiguous nucleic acids derived from a region of the MS4A2 gene that comprises one or more of the polymorphisms shown in Table 1.
  • a diagnostic kit comprises a polynucleotide as defined above.
  • Figure 1 shows the Fc ⁇ RI- ⁇ /MS4A2 genomic structure and sequence polymorphism. Base A of the ATG initiator Met codon of Fc ⁇ RI- ⁇ is denoted nucleotide +1. Dinucleotide repeats are in brackets.
  • Figure 2 shows the Linkage Disequilibrium within Fc ⁇ RI- ⁇ / 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.
  • the present invention utilises known methods of genetic analysis to determine whether a particular subject has, or is predisposed to, an atopic disease.
  • genetic predisposition refers to an increased likelihood that a given subject has or is likely to develop an atopic disease, given the presence of a particular genomic sequence (polymorphism).
  • atopic disease is intended to refer to the related group of diseases, such as, asthma, atopic dermatitis and allergic rhinitis.
  • allele is used herein to refer to variants of a nucleotide sequence.
  • a biallelic polymorphism has two forms; designated herein as "allele 1 " and "allele 2". Diploid organisms may be homozygous or heterozygous for an allelic form.
  • haplotype is used herein to refer to a combination of alleles present on an individual chromosome.
  • polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or subjects.
  • a single nucleotide polymorphism is a single base pair change. Typically, a SNP is the replacement of one nucleotide by another nucleotide at the polymorphic site.
  • dbSNP accession number is given where available (www.ensembl.org); the superscript (a) refers to dinucleotide repeats.
  • the study panels were not genotyped for -3264, -3226, -528, +1798, +11430, +12400.
  • the polymorphisms are shown in brackets. Having identified the SNPs, it will be apparent to the skilled person how to detect polymorphisms for a particular subject, to make a diagnosis. Methods for the detection of a polymorphism are known in the art, and include: polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) (e.g.
  • SSCP Single-stranded conformation polymorphism
  • the method for detecting the presence of a polymorphism comprises: contacting an isolated genetic sample containing the MS4A2 gene, or a portion thereof containing a putative polymorphism, with an oligonucleotide that hybridises to the gene or gene portion if the polymorphism is present; and determining whether hybridisation has occurred.
  • the oligonucleotides can be labelled with a detectable label, e.g. a fluorophore, so that those oligonucleotides that hybridise to the mutated gene can be identified.
  • a preferred method for the identification of the presence of a SNP is to use the LightCycler system (Lohmann et al., Biochemica, 2000; (4): 23-28) developed by Roche Molecular Biochemicals.
  • the LightCycler system enables the amplification and real-time detection of a polynucleotide, allowing accurate quantification.
  • the system permits the detection and genotyping of single nucleotide polymorphisms by utilising a function known as melting curve analysis. During the melting curve analysis the LightCycler instrument monitors the temperature-dependent hybridisation of sequence specific hybridisation probes to single stranded DNA.
  • a genetic sample is contacted with two oligonucleotides designed to hybridise at adjacent sites at the polymorphic region.
  • a ligase reaction is then carried out to ligate those oligonucleotides that are hybridised and the ligated product detected in a subsequent detection step.
  • This method is disclosed in US Patent No.6027889.
  • An alternative detection method is to use what are referred to in the art as "Molecular beacons".
  • Molecular beacons are oligonucleotides designed for the detection and quantification of target nucleic acids.
  • the oligonucleotides usually comprise self-hybridising portions that, in the absence of a target nucleic acid, form a stem-loop structure.
  • a fluorescent moiety and a quencher moiety are attached at each end of the oligonucleotide, and are positioned adjacently when the oligonucleotide is in the stem-loop orientation. Fluorescence is effectively prevented by the quencher moiety in this orientation.
  • the loop portion of the oligonucleotide is complementary to a specific target nucleic acid and, in the presence of the target, hybridisation to the target occurs disrupting the stem loop orientation, separating the fluor and quencher, resulting in an increase in detectable fluorescence.
  • hybridisation probes for use in the LightCycler system, or any hybridisation based system.
  • the design of suitable polynucleotide/hybridisation probes will be apparent to the skilled person.
  • the probes will usually comprise the polymorphic site, e.g. the SNP.
  • the polynucleotides/hybridisation probes may be detectably labelled, e.g. fluorescently labelled, using methods and labels known in the art, e.g. as used in the detection methods referred to above.
  • the polynucleotides/hybridisation probes may be immobilised to a support material, for use in a diagnostic assay.
  • Suitable support materials are known in the art and include, ceramics, plastics, glass and silicon materials.
  • Methods for immobilising polynucleotides to a support material are also known in the art.
  • Polynucleotide array technology are suitable for use in the invention, for screening of biological samples.
  • Arrays that include the desired immobilised polynucleotides can be produced on a customised basis by various companies, including HySeq. In general, the arrays employ immobilised polynucleotide probes that are complementary to target sequences from a biological sample (e.g. from a subject).
  • the target sequence will include a polymorphism as disclosed herein.
  • the polynucleotides to be used as probes in a diagnostic method will usually complete at least an 8, 10, 15, 20 or 50 consecutive nucleic acid sequence derived from the appropriate MS4A2 region, including one or more of the polymorphic sites disclosed herein.
  • Polynucleotides may also be designed to act as primers to amplify polynucleotides that may comprise a polymorphism.
  • One or more polynucleotides may be used to characterise/determine more than one different SNP.
  • linkage analysis The association between the presence of polymorphisms in the MS4A2 gene and atopic disease was identified by studying the correlation between the transmission of genetic markers and the prevalence of atopic disease throughout generations within a family (so-called linkage analysis).
  • Linkage Analysis When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. With loci that are genetic markers, a genetic map can be established, and the strength of linkage between markers and disease states can be calculated and used to indicate the relative positions of markers and genes affecting those disease states.
  • the classical method for linkage analysis is the logarithm of odds (LOD) score method (Morton et al., Am. J. Hum. Genet., 1955; 7: 277-318).
  • Example MATERIALS AND METHODS Physical Mapping Human genomic PAC clones containing Fc Rl- I MS4A2 sequence were identified through hybridisation based library screening using a 690bp cDNA clone, covering exons 2 to 7 of MS4A2, and a 662bp 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.
  • Fc Rl- was located by hybridising the 690bp cDNA probe onto the restriction fragments of the clones.
  • a vectorette PCR technique adapted from Munroe et al. (50) was used to extend the sequence of K ⁇ ster 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 ofthe previously described sequence (13). All sequences were aligned (GAP4 program, STADEN package) to produce 18098 bases of continuous sequence for polymorphism detection, as shown in the attached sequence listing.
  • Panel 2 contained 380 subjects in 67 nuclear and 7 extended pedigrees from the UK, recruited through probands attending hospital clinics with symptoms of asthma or atopic disease (51 ). Phenotvping The same protocols were used to test both panels of families. Prick Skin Tests to House Dust Mite (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 "Skin Test Index (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). A “RAST index (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 ⁇ -
  • 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 (54). Pair-wise D' measurements were made between SNPs from the parental (i.e. unrelated subject) haplotypes and linkage disequilibrium (LD) across the locus was plotted by the GOLD program (55). 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 (56, 57) 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 10bp 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.
  • the ⁇ +6858 polymorphism (E237G (18)) confers the only coding change found.
  • Six of the sequence variants have previously been reported in the literature, ⁇ -211 (originally named -109 (33)), ⁇ +1343 (Rsal-ex2 (14, 27)), the dinucleotide repeat ⁇ +5026 (FCERIB (5, 8)), ⁇ +3934 (33), ⁇ +6858 (E237G (18) and ⁇ +9424 (Rsal-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).
  • 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 ofthe 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 if SNPs in these regions independently contributed to the phenotype.
  • the ⁇ +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 ⁇ +3934*2 cluster is not the only determinant of positive associations with the locus.
  • 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).
  • TF binding sites were altered by the presence of SNPs, using the Matlnspector program (34).
  • IRF-2 Interferon regulatory factor 2
  • Table 4 TFs corresponding to atopy-associated SNPs are shading. SNPs are shown in bold in the consensus recognition sequence.
  • haplotypes also do not suggest that a single SNP is responsible for association of atopy phenotypes to this locus. Although most ofthe 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. These results are not consistent with the haplotype block hypothesis
  • Fc ⁇ RI- ⁇ chain is not essential for Fc ⁇ 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 to 30 fold amplifier of Fc ⁇ RI- ⁇ mediated cell activation signals (12, 22, 41 ).
  • Receptor function may therefore be modified by variation in the level of ⁇ chain expression, or in the level of the recently recognised truncated form, ⁇ ⁇ , which regulates receptor surface expression (38).
  • IRF-2 Interferon regulatory factor 2
  • IRF-2 polymorphisms have been associated with atopic eczema (40) and IRF-2 knockout mice show defects in CD8+ T cells and spontaneous development of an inflammatory skin disease (41 ).
  • 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.
  • Parent-of-origin effects have been observed at other loci influencing allergic disease (8, 42, 43) and in other immunological disorders such as type I diabetes (44), Crohn's disease (45), rheumatoid arthritis (46) and IgA deficiency (47) .
  • the strength of parent-of-origin effects in type I diabetes had also been observed to differ among family collections from different populations (48). No mechanism has been identified for these phenomena.
  • Interaction between maternal and foetal immune systems, and genomic imprinting of disease genes are two possibilities (49). The two regions of increased CpG concentration that we have identified in Fc ⁇ RI- ⁇ provide a potential substrate for epigenetic effects.
  • the content of each of the publications referred to in the specification is incorporated herein by reference. References
  • IRE-bubble PCR a rapid method for efficient and representative amplification of human genomic DNA sequences from complex sources. Genomics, 19, 506-514.

Abstract

Detecting whether a subject has or is predisposed to an atopic disorder can be carried out by determining whether the subject has any of the identified genetic polymorphisms located in the MS4A2 gene.

Description

DIAGNOSIS OF ATOPIC DISORDER
Field of the Invention This invention relates to a diagnostic assay for the detection of a risk of asthma or atopy. Background of the Invention 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 characterised 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 (FcεRI) 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 FcεRI (FcεRI-β oτMS4A2: OMEVI 147138) as modifying the prevalence of atopic disease (5-9). Preferential transmission of atopic diseases from mothers rather than fathers is well recognised (reviewed in (10)), and in several studies linkage has only been observed to alleles derived from the mother (5-8, 11 ). The FcεRI-β protein acts as an amplifying element of the high-affinity \gΕ. receptor response to activation (12) and in addition stabilises the expression of the receptor on the mast cell surface (4). Polymorphism in FcεRI-β is therefore in an ideal position to modify FcεRI signalling in response to allergens. FcεRI-β is composed of seven exons and six introns, spanning approximately 11 Kb (13). Initial sequencing of the coding regions of FcεRI-β detected polymorphisms within exon 6 (L181I and L183V), which is 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 FcεRI-β 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 FcεRI-β 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 ofthe total serum IgE concentration in Caucasian
(28) and Aboriginal Australians (28). The phenotypes with which FcεRI-β 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). The observed associations of FcεRI-β Single Nucleotide Polymorphisms (SNPs) to elevated total and specific serum IgE suggest as yet unrecognised mechanisms influencing IgE synthesis, that lie beyond simple allergen-induced degranulation. 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 FcεRI-β did not show a maternal bias. This suggests that a variable process underlies the maternal effect at this locus. WO02/062946 discloses human and mouse proteins that span the cell membrane at least four times and share high levels of sequence identity with FcεRI -β, CD20 and HTm4. These proteins are stated to reveal a new gene family, designated as MS4A. Although the FcεRI-β gene, designated as MS4A2, is disclosed as being useful in the treatment of atopic disorders, this is in the context of evaluating drugs that modulate the MS4A2 protein function. WO97/08338 discloses the E237G polymorphism in exon 7 of FcεRI-β. A method for diagnosing atopy based upon the identification of this polymorphism is also disclosed. WO95/05481 discloses the identification of specific polymorphisms at amino acids 181 and 183, in exon 6 of FcεRI -β. Van Hage-Hamsten et al, Clin. Exp. Allergy, 2002; 32: 838-842 and Kim et al, Clin. Exp. Allergy, 2002; 32: 751-755 investigate the link between FcεRI-β polymorphisms and allergic responses, including asthma and atopy. Associations between polymorphisms and allegen-specific IgE levels are disclosed. The studies conclude that polymorphisms, in particular E237G, are linked to IgE-mediated histamine release from basophils, allergic responses and atopy. Despite evidence for the importance of the FcεRI-β 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. Summary of the Invention The present invention is based on the finding that polymorphisms within the MS4A2 (FcεRI-β) gene are associated with atopic disease. According to a first aspect ofthe present invention, a method for detecting whether a subject has or is predisposed to an atopic disease comprises: determining the presence in the subject of any of the genetic polymorphisms disclosed in Table 1. According to a second aspect, an isolated polynucleotide useful for diagnosing whether a subject has or is predisposed to an atopic disease, comprises at least 15 contiguous nucleic acids derived from a region of the MS4A2 gene that comprises one or more of the polymorphisms shown in Table 1. According to a third aspect, a diagnostic kit comprises a polynucleotide as defined above. Description of the Drawings The invention is described with reference to the accompanying drawings, wherein: Figure 1 shows the FcεRI-β/MS4A2 genomic structure and sequence polymorphism. Base A of the ATG initiator Met codon of FcεRI-β is denoted nucleotide +1. Dinucleotide repeats are in brackets. Figure 2 shows the Linkage Disequilibrium within FcεRI-β / 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. Description of the Invention The present invention utilises known methods of genetic analysis to determine whether a particular subject has, or is predisposed to, an atopic disease. As used herein, "genetic predisposition" refers to an increased likelihood that a given subject has or is likely to develop an atopic disease, given the presence of a particular genomic sequence (polymorphism). The reference to "atopic disease" is intended to refer to the related group of diseases, such as, asthma, atopic dermatitis and allergic rhinitis. The term "allele" is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms; designated herein as "allele 1 " and "allele 2". Diploid organisms may be homozygous or heterozygous for an allelic form. The term "haplotype" is used herein to refer to a combination of alleles present on an individual chromosome. The term "polymorphism" as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or subjects. A single nucleotide polymorphism (SNP) is a single base pair change. Typically, a SNP is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide, or insertion of a single nucleotide, also gives rise to single nucleotide polymorphisms. The reference to FcεRI-β is used interchangeably with MS4A2. The polymorphisms associated with a predisposition to atopic disease are found within the MS4A2 gene. The SNPs are listed in Table 1. Table 1 Polymorphism bta f Alleles and surrounding sequence Heterozygosity Panel 1 Panel 2 εβ -3383 - CCATTCTAA(C/T)TGGCGCAAG 51.6 45.2 εβ-3264a - TTTTGCGAA(GT)nTCATATCCT 16.0 (n=60) εβ -3226 - GTTGTTTAT(A G)TTTTTTTCT 45.5 (n =20) εβ -2384 - CTGTTGACA(A/G)TTCTCACAA 52.0 45.0 εβ-2381 - TTGACATT(C/T)TCACAAAAC 53.4 45.4 εβ-1964Δ - GGTGGAGTT(TGC/-)GGTGAGCCG 42.9 38.7 εβ -1667 524748 CCAGCTACT(CT)GGGAGGCTG 51.1 46.9 εβ -854 573790 TAATTCTAA(C/T)TTAGGCATA 50.0 48.7 εβ -756 574700 TTCTTGCCA(CT)TGTAAAGAT 3.8 4.8 εβ -528 1441585 ACTAACACA(T/C)ACTCACTCA 8.0 (n= =24) εβ-211 1441586 TAGACTTCT(C/T)AATTTTTCT 55.4 47.2 εβ+1343 - CATGACTAC(G/A)TACATAAAG 49.4 46.2 εβ +1798 - CAGTATTAA(G/C)ATGATATTT 46.8<n=8) εβ+2258 - ATAGTGCAT(A/G)CCTGTAATC 50.9 44.7 εβ+2471 556917 TAAGATCAA(T/A)GGGCATATG 52.3 46.0 εβ +3332 - ACGATTGCA(G/A)TAGAATGAGA 50.8 45.3 εβ+3365 - CGATTCATG(CT)CCTGATGTT 50.0 45.6 εβ+3934 502581 TACCCCAAAT(T/G)TTACCTATG 52.2 46.4 εβ+5026a - GCCTGTGTT(TG)nTATGTGTCA 68.4 68.2 εβ+5565 - GATGGATGT(A/G)CAGAAGGCT 49.6 45.2 εβ +5734 - CAAATGTTT(C/T)CACTTGCCT 48.9 44.3 εβ+6858 569108 ACCCAGGGG(A G)AATGTCTCC 5.1 5.4 εβ+7007 512555 AGATTTATT(C/T)GCCTGATAA 3.9 5.3 εβ +8033 1290426 AATCGCTT(G/C)AACCTGGG 49.4 47.5 εβ +8900 - TATTAGTCA(C/T)AATTAATGA 8.1 7.2 εβ +9424 - TTTATAGTA(C/T)GCCCACCTC 49.7 46.0 εβ +9928 502419 CAGAATGTA(A/G)GGACAATCA 55.1 47.0 εβ +10062 - TTTTTTCTC(T/C)TTGTGAACC 48.0 44.8 εβ+11130 - GTTAGTATC(T/C)CTCTGTGTT 50.9 45.4 εβ+11332 - GCAGTAGCT(G/A)TATTAGGTA 8.4 6.3 εβ +11430 576616 ATTGTGCAT(G/A)TTCAAAAA 4.9 (n= =40) εβ +11664 574704 CTACTTCCA(A/G)ATAAATTGAA 49.4 46.1 εβ +12209 129440 AATGGTGCT(G/A)CCATGTTTT 3.4 5.4 εβ +12400 1286182 GAGGAGAGA(A/A)GATCCACTT 4.9 (n= =40) εβ+12522 - AATCCTATA(C/T)GAGAGTAAG 0.3 1.2 εβ +12841 1286181 AATTAGATC(G/A)AAACAAAAC 49.9 46.0 εβ +12870 1286181 TTTTCACAG(C/T)GCTTTTNTA 48.2 46.8 εβ +13948 1286179 ATGGGTCCA(C/A)ACGGTTCGC 50.6 45.8 Polymorphisms are labelled in Table 1 by their marker position (bp) relative to the first transcribed nucleotide in Fc Rl- (+1 ). The dbSNP accession number is given where available (www.ensembl.org); the superscript (a) refers to dinucleotide repeats. The study panels were not genotyped for -3264, -3226, -528, +1798, +11430, +12400. The polymorphisms are shown in brackets. Having identified the SNPs, it will be apparent to the skilled person how to detect polymorphisms for a particular subject, to make a diagnosis. Methods for the detection of a polymorphism are known in the art, and include: polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) (e.g. Ju et al, Proceeding of the Eleventh International Histocompatibility Workshop and Conference, Vol.2, 1992, pgs 317-319); PCR sequencing; ligase chain reaction (LCR) Abravaya et al., 1995, NAR, 23(4): 675-682); oligotyping using Sequence Specific Primers (SSP) (e.g. Olerup et al., Proceedings of the Eleventh International Histocompatibility Workshop and Conference" Vol. 2, 1992, pgs 315-317); oligotyping using Sequence Specific Oligonucleotide Probes (SSOP) (Tiercy et al., Immunobiology of HLA, Vol. II, pp.248-250, 1987, Springer Verlag, New York); Single-stranded conformation polymorphism (SSCP) (Yap et al., Feb/1992, Trends in Genetics, 8(2):49; and Orita et al., 1989, Genomics, 5: 874-879); and direct sequencing of the 3' flanking region of the HLA-B locus gene (see, e.g. Santamaria etal., Proceedings of the Eleventh International Histocompatibility Workshop and Conference" Vol. 2, 1992, pgs 342-345). The present invention is not limited to any particular method for detecting a polymorphism. In general, the method for detecting the presence of a polymorphism comprises: contacting an isolated genetic sample containing the MS4A2 gene, or a portion thereof containing a putative polymorphism, with an oligonucleotide that hybridises to the gene or gene portion if the polymorphism is present; and determining whether hybridisation has occurred. The oligonucleotides can be labelled with a detectable label, e.g. a fluorophore, so that those oligonucleotides that hybridise to the mutated gene can be identified. A preferred method for the identification of the presence of a SNP is to use the LightCycler system (Lohmann et al., Biochemica, 2000; (4): 23-28) developed by Roche Molecular Biochemicals. The LightCycler system enables the amplification and real-time detection of a polynucleotide, allowing accurate quantification. The system permits the detection and genotyping of single nucleotide polymorphisms by utilising a function known as melting curve analysis. During the melting curve analysis the LightCycler instrument monitors the temperature-dependent hybridisation of sequence specific hybridisation probes to single stranded DNA. In a further preferred method for detecting polymorphisms, a genetic sample is contacted with two oligonucleotides designed to hybridise at adjacent sites at the polymorphic region. A ligase reaction is then carried out to ligate those oligonucleotides that are hybridised and the ligated product detected in a subsequent detection step. This method is disclosed in US Patent No.6027889. An alternative detection method is to use what are referred to in the art as "Molecular beacons". Molecular beacons are oligonucleotides designed for the detection and quantification of target nucleic acids. The oligonucleotides usually comprise self-hybridising portions that, in the absence of a target nucleic acid, form a stem-loop structure. A fluorescent moiety and a quencher moiety are attached at each end of the oligonucleotide, and are positioned adjacently when the oligonucleotide is in the stem-loop orientation. Fluorescence is effectively prevented by the quencher moiety in this orientation. The loop portion of the oligonucleotide is complementary to a specific target nucleic acid and, in the presence of the target, hybridisation to the target occurs disrupting the stem loop orientation, separating the fluor and quencher, resulting in an increase in detectable fluorescence. The use of the molecular beacons approach to the detection of SNPs is disclosed in US 6548254. Using the known sequence information for the MS4A2 gene region, including the known polymorphisms and the novel polymorphisms disclosed herein, it is possible to design hybridisation probes for use in the LightCycler system, or any hybridisation based system. The design of suitable polynucleotide/hybridisation probes will be apparent to the skilled person. The probes will usually comprise the polymorphic site, e.g. the SNP. The polynucleotides/hybridisation probes may be detectably labelled, e.g. fluorescently labelled, using methods and labels known in the art, e.g. as used in the detection methods referred to above. The polynucleotides/hybridisation probes may be immobilised to a support material, for use in a diagnostic assay. Suitable support materials are known in the art and include, ceramics, plastics, glass and silicon materials. Methods for immobilising polynucleotides to a support material are also known in the art. Polynucleotide array technology (DNA chips) are suitable for use in the invention, for screening of biological samples. Arrays that include the desired immobilised polynucleotides can be produced on a customised basis by various companies, including HySeq. In general, the arrays employ immobilised polynucleotide probes that are complementary to target sequences from a biological sample (e.g. from a subject). In the context of the present invention, the target sequence will include a polymorphism as disclosed herein. The polynucleotides to be used as probes in a diagnostic method will usually complete at least an 8, 10, 15, 20 or 50 consecutive nucleic acid sequence derived from the appropriate MS4A2 region, including one or more of the polymorphic sites disclosed herein. Polynucleotides may also be designed to act as primers to amplify polynucleotides that may comprise a polymorphism. One or more polynucleotides may be used to characterise/determine more than one different SNP. The association between the presence of polymorphisms in the MS4A2 gene and atopic disease was identified by studying the correlation between the transmission of genetic markers and the prevalence of atopic disease throughout generations within a family (so-called linkage analysis). Linkage Analysis When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. With loci that are genetic markers, a genetic map can be established, and the strength of linkage between markers and disease states can be calculated and used to indicate the relative positions of markers and genes affecting those disease states. The classical method for linkage analysis is the logarithm of odds (LOD) score method (Morton et al., Am. J. Hum. Genet., 1955; 7: 277-318). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA (19189); David et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987). The following Example illustrates the invention. Example MATERIALS AND METHODS Physical Mapping Human genomic PAC clones containing Fc Rl- I MS4A2 sequence were identified through hybridisation based library screening using a 690bp cDNA clone, covering exons 2 to 7 of MS4A2, and a 662bp 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 Rl- was located by hybridising the 690bp cDNA probe onto the restriction fragments of the clones. A vectorette PCR technique adapted from Munroe et al. (50) was used to extend the sequence of Kϋster 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 ofthe previously described sequence (13). All sequences were aligned (GAP4 program, STADEN package) to produce 18098 bases of continuous sequence for polymorphism detection, as shown in the attached sequence listing. Polymorphism detection Variants within FcεRI-β and the surrounding region were detected by sequencing 12 unrelated British Caucasian individuals. The sequences were analysed for variants using PhredPhrap (http://www.phrap.org). Additional SNPs within FcεRI-β were located on dbSNP (http://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 7 extended pedigrees from the UK, recruited through probands attending hospital clinics with symptoms of asthma or atopic disease (51 ). Phenotvping The same protocols were used to test both panels of families. Prick Skin Tests to House Dust Mite (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 "Skin Test Index (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). A "RAST index (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 εβ-
1964ΔTGC deletion was genotyped by ARMS analysis. Detailed PCR and RFLP conditions are given in Table 1 in supplementary information. Two individuals checked genotypes independently without knowledge of phenotype. The study panels were not genotyped for SNPs εβ-3226, εβ-528, εβ+1798, εβ+11340, εβ+12400 either because the polymorphisms were rare or because they could not be typed by restriction digestion. The εβ-3264 dinucleotide repeat also was not typed in the full sample because of its low information content. The dinucleotide repeat εβ+5026 (FCERIB) was genotyped as described previously (8, 51). Statistical Analysis Tests of association with the quantitative traits were performed in a variance components framework using the QTDT program (52). 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 (53) in which a model including ordered mating types and association including dominance was compared to a model that allowed heterozygotes to be different depending on which parent was being tested (43). Maternal effects were then examined by QTDT (52). It was recognised that the transmission tests were less powerful than the total association model because they only used information from children. The dinucleotide repeat εβ+5026 was tested for association using the multi-allelic option of QTDT. In order to further refine the localisation ofthe 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 (54). Pair-wise D' measurements were made between SNPs from the parental (i.e. unrelated subject) haplotypes and linkage disequilibrium (LD) across the locus was plotted by the GOLD program (55). 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 (56, 57) 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 10bp 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 Matlnspector 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 basepairs of random DNA sequence. The matrix similarity is calculated as described (34). 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. RESULTS Polymorphism within FcεRI-β (MS4A2) and surrounding seguence A physical map of the locus was constructed and sequenced, to form a contiguous 18,098 bp region. A total of thirty-eight sequence variants were detected in the sequence. The nature of the sequence change and its position relative to the first nucleotide of the FcεRI-β 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, εβ- 1862ΔTGC, involved a 3 base deletion. The εβ+6858 polymorphism (E237G (18)) confers the only coding change found. Six of the sequence variants have previously been reported in the literature, εβ-211 (originally named -109 (33)), εβ+1343 (Rsal-ex2 (14, 27)), the dinucleotide repeat εβ+5026 (FCERIB (5, 8)), εβ+3934 (33), εβ+6858 (E237G (18) and εβ+9424 (Rsal-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 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 εβ+3264 dinucleotide repeat was of low heterozygosity and was also not typed (Table 1). Linkage Disequilibrium within FcεRI-β (MS4A2) The regional distribution of linkage disequilibrium (LD) within FcεRI-βwas seen to be irregular, but approximately distributed into proximal and distal islands (Figure 2). Three SNPs in the promoters of the gene (εβ-3383, εβ-2384 and εβ-2381 ) showed strong LD with each other but little LD with the rest of the gene. The adjacent εβ-1964Δ 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 εβ+8033 (minor allele frequency 0.47) and εβ+12209 (minor allele frequency 0.05) showed low levels of LD with most markers. Associations with atopy phenotypes 62% of the subjects in Panel 1 were atopic and 18% were asthmatic, compared to 67% atopic and 45% asthmatic in Panel 2. The mean loge IgE concentration was 3.87; variance (σ2) = 2.76 in Panel 1 and 4.18; σ2 = 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; σ2 = 9.1 in Panel 1 and 2.7; σ2 = 12.7 in Panel 2; and mean RASTI 2.1 ; σ2 = 6.8 in Panel 1 and 1.8; σ2 = 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. Table 2
Positive results of allelic association tests in two panels of subjects The results of association testing to all alleles (clear columns) are compared to the results of testing to maternally derived alleles (shaded columns). Panel 1 were derived from a general population sample and Panel 2 were recruited through a proband with atopic asthma.
Figure imgf000015_0001
a PSTI with RASTI included as a covariate b only maternally derived alleles tested c dinucleotide repeat 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 ofthe 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 if SNPs in these regions independently contributed to the phenotype. The εβ+3934 SNP (P=0.006) was first included as a covariate in the QTDT analysis: the most significantly associated remaining SNP, εβ+9424 (p=0.004), was then included; then εβ+5565 (P=0.06); then εβ+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 (Figure 2). We therefore generated an extended haplotype of all four SNPs. εβ+1343*2 / εβ+3934*1 / εβ+5565*1 / εβ+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 εβ+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 εβ+3934*2 cluster is not the only determinant of positive associations with the locus. Table 3 εβ+1343 εβ+3934 εβ+5565 εβ+9424 HAPLOTYPE Freq S (P) Variance *2 *1 *1 *1 0.54 7.93 (0.005) -1.3% *1 *2 *2 *2 0.37 4.44 (0.035) +0.6% *\ *\ *\ *\ 0.03 2.65 (0J0) +0.5% *2 *2 *2 *2 0.02 3.83 (0.05) +0.7% *2 *2 *1 *1 0.004 6.44 (0.01) +1.3% 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 , εβ+3934, P=0.00004; step 2, εβ+5734, P=0.0002; step 3, εβ+1343, P= 0.0023; step 4, εβ-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 -1748 and ending at -1608 (Mott Island Score (MIS) = 201 ; P = 0.0079), and the second beginning at +10578 and ending at +10814 (MIS = 260; P = 0.0003))(Figure 1). The first CpG concentration (MM ) contained the εβ-1667 SNP, which did not show convincing associations with either phenotype. However, the second concentration (MI2) approximated the highly maternally associated εβ+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 (Figure 1). A reverse promoter (R) was identified between +9637 to +9387. The sequence was examined for potential transcription factor (TF) binding sites that were altered by the presence of SNPs, using the Matlnspector program (34). Interferon regulatory factor 2 (IRF-2) sites are identified in two regions with significantly associated SNPs, and a number of OCT-1 sequences are observed in the F1 and F2 promoters (Table 4). Table 4 TFs corresponding to atopy-associated SNPs are shading. SNPs are shown in bold in the consensus recognition sequence.
Matrix RE Start End Strand Matrix sim. Consensus recognition sequence OCT1.06 0.04 -3392 -3381 (+) 0.819 accattctAATT OCT1.06 0.04 -2384 -2373 (-) 0.805 tttgtgagAATT 0.04 -863 -852 (+) 0.814 ctaattctAATT EVI1.06 0.02 1797 1805 (+) 0.822 acaTGATat HNF4.01 0.03 2464 2477 (+) 0.920 agatCAAAgggcat OCT1.06 0.04 2469 2480 (-) 0.804 catatgccCATT <0.01 3929 3940 (+) 0.800 caAATTttacct <0.01 5730 5742 (-) 0.860 ggcaagtGAAAac
"oLpi.όi 0.03 6843 6864 (-) 0.847 gacattTCCCctgggtcttcca OCT.01 0.05 7001 7013 (-) 0.851 tcAGGCaaataaa BRN3.01 <0.01 8899 8912 (+) 0.861 ACAAttaatgaaat ξ W <0.01 10054 10066 (-) 0.803 acaaagaGAAAaa RORA2.01 0.01 12830 12842 (+) 0.868 gtaattaGATCaa
DISCUSSION This study is likely to have identified all common polymorphisms in the genomic sequence of the FcεRI-β gene and upstream and downstream DNA. The sequencing of 12 unrelated individuals has given 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 (35) 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. The stepwise analyses indicate that the clusters represent independent effects. 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. Although most ofthe 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. These results are not consistent with the haplotype block hypothesis
(36, 37), and the typing of a limited number of SNPs from this gene might lead to misleading results from association testing. This study identified only one coding polymorphism, εβ+6858 (E237G) and this does not have functional effects on FcεRI receptor expression or signalling (22). The FcεRI-β chain is not essential for FcεRI function and does not possess autonomous cell activation capacity, but it augments the surface expression of the receptor (22) and acts as a 12 to 30 fold amplifier of FcεRI-γ mediated cell activation signals (12, 22, 41 ). Receptor function may therefore be modified by variation in the level of β chain expression, or in the level of the recently recognised truncated form, βτ, which regulates receptor surface expression (38). The study has identified several potential transcription factor (TF) binding sites that are affected by SNPs. In silico prediction of TF binding sites is non- conservative, and any predictions by Matlnspector and similar programs should be the prelude to functional studies (39). Nevertheless, it may be of interest that Interferon regulatory factor 2 (IRF-2) sites are identified in two regions with significantly associated SNPs. Modifications of these binding sites may therefore be useful in the treatment of asthma. 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 (40) and IRF-2 knockout mice show defects in CD8+ T cells and spontaneous development of an inflammatory skin disease (41 ). 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 categorised 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 FcεRI 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, 42, 43) and in other immunological disorders such as type I diabetes (44), Crohn's disease (45), rheumatoid arthritis (46) and IgA deficiency (47) . The strength of parent-of-origin effects in type I diabetes had also been observed to differ among family collections from different populations (48). No mechanism has been identified for these phenomena. Interaction between maternal and foetal immune systems, and genomic imprinting of disease genes are two possibilities (49). The two regions of increased CpG concentration that we have identified in FcεRI-β provide a potential substrate for epigenetic effects. The content of each of the publications referred to in the specification is incorporated herein by reference. References
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Claims

1. A method for detecting whether a subject has or is genetically predisposed to an atopic disease, comprising: determining the presence in the subject of one or more of the genetic polymorphisms shown in Table 1.
2. A method according to claim 1 , comprising contacting an isolated genetic sample containing the MS4A2 gene, or a portion thereof containing a putative polymorphism, with an oligonucleotide that hybridises to the gene or gene portion if the polymorphism is present; and determining whether hybridisation has occurred.
3. A method according to claim 2, wherein the oligonucleotide is detectably labelled.
4. A method according to claim 3, wherein the label is a fluorophore.
5. A method according to any preceding claim, wherein the genetic polymorphism is not one of those referred to as εβ-211 , εβ+1343, εβ+5026, εβ 3934, εβ+9424 and εβ+6858.
6. A method according to any preceding claim, wherein the presence of two or more polymorphisms is determined on either a single allele or two alleles.
7. An isolated polynucleotide useful in diagnosing whether a subject has or is predisposed to an atopic disease, comprising at least 15 contiguous nucleic acids and hybridizing under highly stringent conditions to a region of the gene MS4A2 that comprises a polymorphism shown in Table 1 , or its complement.
8. Use of a polynucleotide according to claim 7, for determining whether a subject has or is predisposed to an atopic disease.
9. A diagnostic kit comprising in a discrete compartment a polynucleotide as defined in claim 7.
PCT/GB2004/003518 2003-08-14 2004-08-16 Diagnosis of atopic disorder WO2005017505A2 (en)

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EP1876244A1 (en) * 2005-04-04 2008-01-09 Asubio Pharma Co., Ltd. Method of relative risk for the onset of atopic dermatitis by gene single nucleotide polymorphism analysis
EP1876244A4 (en) * 2005-04-04 2009-09-23 Asubio Pharma Co Ltd Method of relative risk for the onset of atopic dermatitis by gene single nucleotide polymorphism analysis
US8071307B2 (en) 2005-04-04 2011-12-06 Asubio Pharma Co., Ltd. Method of detecting relative risk for the onset of atopic dermatitis by gene single nucleotide polymorphism analysis

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