SUSCEPTIBILITY GENE FOR AUTOIMMUNE DISEASE

Abstract: A method is described for determining, in a human subject, a predisposition to autoimmune thyroid disease such as autoimmune hypothyroidism or Grave's disease. The method comprises determing the genotype of the human subject at the -590 position of the promoter region of the Il-4 gene.

Description: SUSCEPTIBILITY GENE FOR AUTOIMMUNE DISEASE The present invention relates to a novel association between a known polymorphism in the promoter region of the human 11-4 gene and the autoimmune thyroid diseases Grave's disease (GD) and autoimmune hypothyroidism (AIH) . Autoimmune thyroid disease, comprising predominantly of Graves' disease (GD) and autoimmune hypothyroidism (AIH) , is common, affecting approximately 2% of women and 0.2% of men. The aetiology of the disease remains unknown but appears to involve a complex interplay of multiple genetic and environmental influences. Evidence for the role of genetic factors is shown by the increased incidence of the disease within families and by twin studies. For well defined Grave's disease the concordance rate in monozygotic twins is reported as 22% and 0% for the dizygotic twins (Brix et al 1998). Like other autoimmune disorders, the human leukocyte antigen (HLA) class II genes contribute to susceptibility and studies also suggest a role for the cytotoxic T lymphocyte associated-4 (CTLA-4) gene (Brix et al 1998b) . However each of these candidate genes is likely to contribute no more than 5% to the overall genetic susceptibility. Other potential susceptibility loci have recently been identified by linkage analysis on chromosomes X (Barbesino et al 1998) , 14q31 (Tomer et al 1998) and 20qll (Tomer et al 1998) but await confirmation. Genome screening using multiple microsatellite markers is now being used for the detection of susceptibility loci in complex human diseases. Results from such analyses have produced promising results for the related autoimmune disease type 1 IDDM (Davies Nature 1994, Hashimoto Nature 1994) . An alternative and complimentary approach examines known polymorphisms in candidate genes, chosen for their role in the pathophysiology of the disease. This has been very effective in other diseases such as Alzheimer's disease (Roses 1996) and Human Immunodeficiency Virus infection (Samon et al 1996) . Thus case-control association studies provide a valuable tool for investigating potential susceptibility genes and also genes important for disease progression. Cytokines participate in.the induction and effector phases of the immune and inflammatory response and are therefore likely to play a critical role in the development of autoimmune thyroid disease. Intrathyroidal inflammatory cells and thyroid follicular cells have been shown to produce a variety of cytokines including Il-lα, IL-lβ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, ' tumour necrosis factor (TNFα) and interferon γ (IFNγ) (Ajjan 1996, 1997) . The cytokine network is complex, with cytokines having both diverse and overlapping functions, ' including effects which are promoted or inhibited by other cytokines. Cytokine secretion profiles can be considered a-s either pro- or anti-inflammatory, or .alternatively, on the basis of the animal model as either T helper cell type 1 (Thl) responses promoting cell mediated immunity (IL2 and IFNγ) or T helper cell type 2 (Th2) responses promoting humoral immunity (IL4, IL5, 116, IL10 and IL13) (Mosman 1989). Despite attempts to classify autoimmune thyroid disease as a classical Thl or Th2 mediated disease.no clear conclusions can be drawn, and a mixed Thl/Th2 response is seen in both GD and AIH (reviewed in Ajjan et al 1996) . Cytokines in the thyroid gland also have a role in regulating antigen presentation and lymphocyte trafficking by enhancing expression of HLA class II and ' adhesion molecules on thyroid follicular cells -■ (Ajjan 1996) . In thyroid associated ophthalmopathy, cytokines (IL-lα, TNFα, I:FNγ) are pathogenic, promoting inflammation and fibroblast proliferation leading to the accumulation of glycosaminoglycans (Natt & Bahn 1997) . Polymorphisms in genes encoding these crucial immunomodulatory molecules may result in an altered level of expression and hence must be considered important candidate genes for autoimmune disease susceptibility and severity. Since cytokines interact functionally the present inventors have examined the contribution of several candidate genes with potential immunoregulatory . roles in autoimmune thyroid disease. The candidate genes were selected for investigation if their gene product was likely to be important in the regulation of the cellular or humoral immune response, and if a single nucleotide polymorphism (SNP) within the gene was amenable to genotyping. Il-lα, IL-lβ, IL-1 receptor antagonist (IL-1RA) and 11-1 receptor 1 are closely linked genes on chromosome 2ql3. IL-lα and Il-lβ are pleiotropic cytokines with primarily proin lammatory effects including stimulation of IL-2 and 11-6. Both IL-lα and Il-lβ, as well as the naturally occurring IL-1RA, act via the 11-1 receptor 1. Hence disruption of the balance between IL-lα, IL-lβ, ILIRA and their receptor may result in disease. An IL-lα polymorphism (position -889) has been associated with juvenile rheumatoid arthritis (McDowell 1995 Arthritis & Rheumatism, Ploski 1995) and a functional IL-lβ polymorphism (exon 5, +3962) (Pociot et al 1992. Euro J Clin Invest) has been associated with IDD (Pociot 1992, 1994). Variation in the IL-1RA gene (exon 2) has been associated with a variety of autoimmune disorders including SLE (Blakemore) , ulcerative colitis (Mansfield 1994), IDDM (Blakemore- 1996, Mandrup- . Poulsen et al 1994) as well as autoimmune thyroid disease. An initial association was seen with GD but not Hashimoto's thyroiditis (Tarlow et al 1993) although this finding has not been replicated (Cuddihy and Bahn 1996) . An Il-l receptor 1 polymorphism (position 1970) (Bergholdt et al 1995) has also been investigated by the present inventors. The IL-4 gene is clustered with the genes for IL- 3, IL-5, IL-13 and granulocyte macrophage-colony stimulating factor (GM-CSF) on chromosome 5q22-31 (van Leeuwen) . IL-4 mediates the humoral immune response and a polymorphism (position -590) has been associated with asthma (Rossenwasser 1995) . Also associated with atopy is variation in the IL-4 receptor gene (nucleotide 1902) (NEJM 1997). IL-6 (chromosome 7p21- 15) is a key inflammatory cytokine and elevated systemic levels are seen in many conditions including autoimmune thyroid disease (Ajjon 1996) . Transgenic mice overexpressing IL-6 produce excessive IgGl (Suematsu 1989) which is the thyrotrophin (TSH). receptor antibody isotype that is pathogenic in Graves' disease ( eetman & Mc 1994) . Variation in the promoter region of the IL-6 gene has been associated with juvenile rheumatoid arthritis (Fishman et al 1998) . IL-10 (chromosome lq31-32) enhances B cell proliferation and antagonises the Thl cytokine response. An IL-10 polymorphism (position -1082) resulting in reduced IL-10 levels (Turner et al 1997) has been associated with SLE (Lazarus 1997) and rheumatoid arthritis (Hajaar 1998) . The inventors also examined the haplotype resulting from three SNPs (positions -1082, -819, - 592) in IL-10. Finally ' the ' pleiotropic cytokine, transforming growth factor beta (TGFβ) , plays an important mόdulatory role in cellular growth and differentiation, including an inhibitory role in B cell maturation. 'Several polymorphisms of the TGFβ gene (Cambien et al 1996) on chromosome 19q31 were also evaluated. To investigate whether variability in these immunoregulatory genes may influence -disease susceptibility or severity, the present inventors carried out a case control association study on the known polymorphisms. . Polymorphisms in the genes for IL-lα, IL-lβ, IL-1 receptor antagonist, IL-1 receptor 1, IL-4, IL-4 receptor, IL-6, IL-10 and transforming growth factor beta were investigated in this study. Genotyping was performed using the polymerase chain reaction and sequence specific primers (PCR-SSP) . Analysis of the results of this study indicated a reduced frequency of the variant λ t' allele in the IL- 4 promoter polymorphism (position -590) in the patients with Graves' disease, and the entire patient group (GD and AIH) compared with the control group (Pc=0.00004 and <0.00001 for ' GD and all patients respectively) . This was reflected in a reduction in the heterozygote genotype in the patient groups compared to the controls ( Λ c/.t' heterozygotes GD 12%; Pc=0.06, OR 0.4 (0.2-0.7); all patients 11%; Pc=0.008, OR 0.4 (0.2-07); control subjects 23%). In a first aspect the invention provides a method of screening a human subject. for predisposition to autoimmune thyroid disease, which method comprises determining the genotype of the human subject at the -590 position of the promoter region of the IL-4 gene. Unless otherwise defined, ' all medical and scientific terms used herein have the ordinary meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. ' Accordingly, the term Λ autoimmune thyroid disease' should be taken to encompass both Graves' disease (GD) and autoimmune hypothyroidism (AIH) . In the context of this application the -590 position of the IL-4 promoter is numbered relative to the translation initiation codon, according to the numbering system of Rossenwasser, 1995. The complete nucleotide sequence of the chromosomal gene for human IL-4 is given by Aral et al. Journal of Immunology, 142: 274-282. The process of "determining the genotype of a human subject at the -590 position of the promoter region of the IL-4 gene" may advantageously comprise screening for the presence or absence in the genome of the subject of both the IL-4 promoter -590 Λ t' allele and the IL-4 promoter -590 Λ c' allele or may comprise screening for the presence or absence of either individual allele. The IL-4 -590 promoter polymorphism has been shown to be functional, with increased transcriptional activity attributed to the variant c' allele (Rossenwasser, 1995) . However, it is also possible that the novel association is due not to the IL-4 gene but to another gene in linkage disequilibrium. Accordingly, the invention further provides a method of screening a human subject for predisposition to autoimmune thyroid disease, which method comprises determining the genotype of the human subject at one or more polymorphic loci in linkage disequilibrium with the IL-4 -590 promoter polymorphism. The genes for IL-3, IL-5, IL-13 and GM-CSF .are all in close proximity to the IL-4 gene and thus may be candidate susceptibility genes. Of these, IL-13 would be an attractive candidate because of the major role it plays in the cytokine network (Kelso, 1998) . Given that both Grave's disease and autoimmune hypothyroidism are likely to be polygenic disorders, possibly sharing a number of susceptibility genes, it is also within the scope of the invention to perform genotyping of the IL-4 -590 ' promoter polymorphism in conjunction with genotyping at other polymorphic loci shown to be associated with • susceptibility to ' autoimmune thyroid disease, for example as part of a panel of screens for possibly up to 10 different polymorphic loci. This might involve two or more polymorphic loci in a single gene or polymorphic loci present in different genes, each of which contributes to the overall genetic susceptibility. In a further aspect the invention provides a method of establishing the or any genetic basis for autoimmune thyroid disease the symptoms of which are manifested in a human subject, which method comprises determining the genotype of said human subject at the -590 position of the promoter region of the IL-4 gene or at one or more polymorphic loci in linkage disequilibrium with the IL-4 -590 promoter polymorphism. The step of determining the genotype of a human subject at the -590 position of the IL-4 promoter (referred to herein as ^genotyping' ) can be carried out using any of the methodologies known in the art and it is to be understood that the invention is in no way limited by the precise technique used to perform genotyping. In a preferred embodiment, genotyping is carried out by performing PCR using allele-specific primers, a technique known in the art as PCR-SSP. In a specific embodiment, exemplified herein, PCR analysis of the - 590 c/t polymorphism is performed on purified genomic DNA using the Λ c' allele forward primer 5'- CTAAACTTgggAgAACATTgTC or the Λ t' forward allele primer 5' -CTAAACTTgggAgAACATTgTT in conjunction with the consensus reverse primer 5' -CACTTggggCCAATCAgCA. Using these primers the predicted amplicon size for both the Λ c' allele and the Λ t' allele is 447bp. The presence of an amplification product in PCR reaction using the Λ c' allele primer and the consensus primer indicates the presence of at least one -590 Λ c' allele in the .genome of the subject,- whereas the presence of an amplification product in PCR.reaction using the λ t' allele primer and consensus primer indicates the presence of at least one -590 Λ t' allele in the genome of said subject. Thus, the three possible genotypes, cc r ct or tt, can be easily distinguished. Further techniques are known in the art for the scoring of single nucleotide polymorphisms (see review by Schafer, A. J. and Hawkins, J. R. in Nature Biotechnology, Vol 16, pp33-39 (1998) including mass spectrometry, particularly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, se Roskey, M. T. et al., 1996, PNAS USA, 93: 4724-4729), single nucleotide primer extension (Shumaker, J. M. et al . , 1996, Hum. Mutat., 7: 346-354; Pastinen, T. et al . , 1997, Genome Res., 7: 606-614) and DNA microchips/microarrays (Underhill, P. A. et al., 1996, PNAS USA, 93: 196-200). The known techniques for scoring polymorphisms are of general applicability and it would therefore be readily apparent to persons skilled in the art that the known techniques could be adapted for the scoring of any single nucleotide polymorphism shown to associated with autoimmune thyroid disease. As would be readily apparent to those skilled in the art, genotyping is generally carried out on genomic DNA prepared from a suitable tissue sample obtained from the subject under test. Most commonly genomic DNA is prepared from a sample of whole blood, according to standard procedures which are well known in the art. The present invention will be further understood with reference to the following non-limiting example: Example 1 Subjects The patient cohort comprised 215 Caucasoid patients with autoimmune thyroid disease (135 GD (111. female) and 77 AIH (67 female) recruited from the Endocrinology clinics in Oxford and Sheffield. GD was defined by the presence of hyperthyroidism and a diffuse goitre, supported by the presence of either thyroid antibodies (peroxidase and/or thyroglobulin) or thyroid eye disease. AIH was diagnosed by the presence of primary hypothyroidism and positive thyroid antibodies with or without a goitre. Information was also obtained on age at diagnosis, size of goitre, presence of other autoimmune disease, and for GD, severity of ophthalmopathy and relapse rates. The study was approved by the respective local ethics committees and all subjects gave written informed consent. The control population comprised 101 Caucasoid cadaveric renal allograft donors. The representative nature of this control population of the general Caucasoid population has previously been shown in HLA genotyping reports (Bunce et al 1996 Tissue Antigens) . Genotyping methodology DNA was extracted from lOmL EDTA blood using the Puregene kit (Gentra Systems, Minneapolis, USA) . All PCR primers were designed with allele specificity determined by the terminal 3' nucleotide. For detection of two or more closely related polymorphisms within the same gene, forward and reverse allele- specific primers were used ( Λ PCR-haplotyping' ) (Lo 1992) thus minimising the number of PCR reactions necessary and formally identifying the cis/trans orientation of the alleles. Primers and concentrations used for IL-lα (-889t/c) , IL-lβ (+3962 t/c), -IL-4 (- 590c/t), IL-6 (+3247a/g) IL-10 (-1082a/g, -819c/t, - 592 c/a) and TGFβ (-880g/a, - 509c/t, aalOL/P, aa263T/I) have previously been described (Mullighan & -Marshall 1999). Primers for IL-lβ (-511 c/t) , IL-1RA, IL-1 receptor 1, and IL-4 receptor are listed in Table 1. To confirm adequate DNA amplification all reaction mixes also contained control primers. Further details of the PCR-SSP methodology including PCR amplification and gel electrophoresis have been published previously (Bunce 1995) . Briefly, DNA was amplified in 13μl reaction mixtures consisting of 67mM Tris base pH 8.8 ; 16.6 nM ammonium sulphate; 2mM magnesium chloride; 0.01% v/v Tween 20; 200mM each of dATP, dCTP, gGTP and dTTP; between 0.1 and O.Olμl DNA;. ' and 0.1875 units of Taq polymerase (Advanced Biotechnology, London, UK) . Primer concentrations were optimised for each reaction and are listed in Table 1. Reaction mixtures were dispensed under lOμl of mineral oil (Sigma, UK) in 96 well PCR plates (Advanced Biotechnology) . PCR plates were sealed with Thermowell sealers (Costar, High Wycombe, UK) and dipped in mineral oil to improve plate to block contact. DNA samples were amplified in GeneAmp PCR system 9600 (Perkin-Elmer Corporation) or in MJ Research PTC-200 thermal cyclers with cycling parameters as follows: one minute at 96°C followed by 5 cycles of 96°C for 25 seconds, 70°C for 45 seconds and 72°C for 45 seconds, followed by 4 cycles of 96°C for 25 seconds, 55°C for 60 seconds and 72°C for 120 seconds. Following PCR, 5μl of loading buffer, consisting of 0.25% Orange G, 30% v/v glycerol and 0.5x TBE buffer (89mM Tris base, 89mM boric acid, 2mM EDTA, pH8.0), was added to each reaction mix. PCR products were electrophoresed in 1.0% agarose gels containing 0.5mg/ml ethidium bromide, for 30-35 minutes at 15V/cam mechanism in 0.5x TBE buffer, visualised with UV illumination and photographed with a Polaroid land camera. Statistical analysis Allele frequencies were compared between- the patient groups and the control group using the binomial distribution probability. Genotype frequencies were also compared between the entire patient group and the control group, and between the subgroups of GD and AIH compared to the control group using the genotype relative risk method of Lathrop (Lathrop M. (1983) Tissue Antigens. 22: 160-166). P values were corrected for the number of independent comparisons of haplotype or allele variants that were made (12) and a corrected P (Pc) value of less than 0.05 was considered significant. Results Fifteen polymorphisms in 9 genes were analysed in both the patients and control subjects. The three polymorphisms in the IL-10 gene and the two in TGFβ (- 800 and -509) were haplotyped (see methods) . All control allele frequencies were in Hardy-Weinberg equilibrium. The major finding was a striking reduction in the frequency of the IL-4 (-590) variant t' allele in the patients with GD and the overall patient group (GD and AIH) compared to the control group (P exact =0.00003, Pc=0.0004 (GD) and P exact <°- 00001, Pc<0.00001 (GD and AIH) (Table 2)). For the patients with AIH a similar trend was seen but this was not significant when corrected for the number of comparisons made (P exact = 0 " 006, Pc=0.07). Genotype analysis showed that the reduction in the Λ t' allele frequency resulted in less IL-4 heterozygote genotypes in the patients with GD compared to controls (P=0.005, Pc=0.06, odds ratio (OR) 0.4, 95% CI (0.2 to 0.7) which was also seen when all patients were compared with- controls (P=0.0007, Pc=0.008, OR 0.4 (0.2 to 0.7)) (Table 3). Again a similar tendency was seen for the patients with AIH (P=0.01, Pc=0.11) but this was not significant when corrected for the number of observations. Allele and genotype frequencies for ' the other gene polymorphisms did not differ significantly between the patients and control groups (data not shown) . Further- subgroup analysis was performed and no significant association ' s of polymorphisms were detected with age of onset of disease, size of -goitre, recurrent GD, thyroid eye disease or the presence of other autoimmune disease (data not shown) . Discussion This study provides the first evidence for a genetic association between autoimmune thyroid disease and the IL-cytokine gene. This association is due to a decreased prevalence of the variant Λ t' allele in the patients with GD, resulting in less Λ c/t' heterozygote genotypes, compared to controls. In addition, in the AIH group a similar trend was seen although these results were not significant when multiple comparisons were taken into account. IL-4 is a key cytokine in immune regulation. It is produced by T cells, mast cells and eosinophills and causes proliferation of IgE and IgG secreting B cells. It also stimulates the expression of HLA Class II antigens via STAT6 (Kelso-1998) and opposes the Thl cell inflammatory response. Indeed, IL-4 is considered as the pivotal cytokine polarising the immune response towards a Th2 cell response and although the initial trigger for IL-4 production remains unknown, genetic variation is likely to play role (Romagnani 1996) . The IL-4 polymorphism investigated in this -study is a Λ c' to Λ t' base change in the promoter region of IL-4 at position -590 (Borish et al 1994) . This. promoter polymorphism appears to be functional with increased transcriptional activity attributed to the variant allele (Rossenwasser, 1995) . Association of the polymorphism with asthma has been demonstrated (Rossenwasser et al 1995, alley et al 1996, Noguchi et al 1998) although evidence relating it to increased IgE levels in asthmatics remains controversial (Rossenwasser et al 1995, Walley, Noguchi) . These results, together with the observation that a gain of function polymorphism of the IL-4 receptor is associated with atopy. (NEJM 97), provides evidence that IL-4 is an important mediator of allergic disease. The mechanism by which IL-4 is involved in the development of autoimmune thyroid disease, particularly GD, requires explanation. T-cell activation is believed to be the key event in the initiation of autoimmune thyroid disease (Weetman & McGregor) and thus cytokines are likely to be intimately involved in the process. Organ-specific autoimmune diseases have been regarded as Thl type diseases (Romagnani), however an exclusive response of a single T cell subset rarely, if ever, exists in vivo (Allen 1997) . Early studies of intrathyroidal lymphocytes from patients with GD and Hashimoto' s thyroiditis (Del Prete 1989) as well as retroorbital tissue from patients with thyroid associated ophthalmopathy (DeCarli 1993) revealed clear cut Thl cytokine responses. Not surprisingly, further studies in autoimmune thyroid disease have identified both Thl and Th2 cytokine profiles (reviewed in Ajjon 1996) . Indeed, such attempts to categorise both autoimmune and infective diseases into distinct Thl or Th2 cell responses is difficult and the whole paradigm of Thl versus Th2 in human disease has been questioned (Kelso 1995) , reiterating the complex nature of the cytokine pathways . The patients with autoimmune thyroid disease in this study exhibited .a lower prevalence of the variant Λ t' allele than the controls which may reflect a overall lower activity of IL-4. This would direct the immune response away from production of IgE and favour , the inflammatory response mediated by Thl cells. In humans Thl cytokines stimulate production of an IgGl isotype response (Abbas AK, Murphy KM and Sher A (1996) Functional diversity of helper T lymphocytes. Nature. 383: 787-793). The pathogenic thyrotropin receptor autoantibodies seen in GD (and sometimes in AIH) are predominantly IgGl (Weetman 1990a, b) . Similarly, IgGl isotypes are prevalent amongst thyroglobulin and peroxidase antibodies (Weetman 1989, Kuppers RG 1993) . Hence in autoimmune thyroid disease, lower IL-4 activity may result in a propensity to develop IgGl autoantibodies along with polarising the immune response towards cell mediated immunity. Cytokines mediating cellular immunity, such as IFNα and IL-2, are known precipitants of autoimmune thyroid disease in humans when given exogenously (Ajjan 1996). Furthermore, in experimental animals there is evidence that IL-4 inhibits organ-specific autoimmune disease. , A reduced incidence of insulin dependant diabetes (IDDM) is seen following IL-4 administration to non- obese mice (Rapport M et al 1993) and 11-4 ameliorates experimental allergic encephalitis (Racke et al 1994) . For GD, the frequency of the variant 't' allele was highly significant and associated with small confidence intervals (OR 0.4 (0.2 to 0.7). While not directly comparable, these results are in keeping with previously identified candidate genes for GD, such as HLA DR3 and CTLA-4 (relative risks of 2 to 5 and 2 to 3, respectively (Brix 1998)). It is of interest that a similar trend for an association of the IL-4 polymorphism was seen in the group AIH, compared to the group with GD. There is certainly evidence suggesting that the two disorders are . closely related and may represent two ends " of a spectrum of autoimmune thyroid disease phenotype. Thus the two diseases commonly cluster within the same family (Aho et al 1983) , monozygotic twins are described where one has GD and the other Hashimoto's thyroiditis (Ilicki et al 1990) and clinically an individual may fluctuate between GD and AIH. The pathogenesis of the two disorders is also related, both being associated with antibodies to thyroid tissue and exhibiting some similarities in immune function (Ajjon et al 1996) . The lack of a significant association after Bonferroni correction for the group with AIH may be because fewer individuals with AIH were genotyped than those with GD (type 1 error) . The other cytokine gene polymorphisms evaluated in this study did not reveal any significant associations with autoimmune thyroid disease. It is relevant to clarify that negative results, whilst important, do not necessarily preclude a role for these genes and the power of the study to detect an association must be considered. For a representative rare allele with a frequency of 0.2 in the control population, this study had 94% power to detect an increase/decrease in the rare allele at an odds ratio of 2.0 and a significance level of P=0.05 for the combined patient group with autoimmune thyroid disease (215 patients) . For GD alone (138 patients) and AIH (77 patients) the power was 90% and 81% respectively. .To identify genes exerting a minor effect, or influencing clinical phenptype, larger study cohorts are required, particularly when several candidate genes are evaluated and adjustments for multiple comparisons are necessary, This study has demonstrated a significant association of IL-4 genotype with susceptibility to autoimmune thyroid disease. This suggests that variation in amplification and regulation of immune responses within the thyroid determine clinical disease. 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Table 1 PCR-SSP primer specificities and sequences Gene Allele Sense primer Cone Annealing Antisense primer polymorphism position (5' to 3') (μM) position (5' to 3') [GenBank accession No.] (ref) IL-1β -511 c CTCATCTggCATTgATCTgg 3.4 1226-1245 ggTgCTgTTCTCTgCCTCg [X04500] (49) -511 1 CTCATCTggCATTgATCTgg 3.4 1226-1245 ggTgCTgTTCTCTgCCTCA IL-1 RA +111001 CCTTCATCCgCTCAgACAgT 0.68 11081-11100 TgACgCCTTCTgAGGGTC [X64532] (50) +11100 c CCTTCATCCgCTCAgACAgC 0.68 11081-11100 TgACgCCTTCTgAGGGTC IL-1 receptor 1 +970 c CCAgCCTggATTTgTCCgg 3.4 700-718 CAgTggTCgAgTCTgCAg [U14179] (51) +970 1 CCAgCCTggATTTgTCCgg 3.4 700-718 CagTggTCgAgTCTgCAA IL-4 receptor +1902 a CAgTCCTCTggCCAgAgAg 5.1 1221-1239 CACCgCATgTACAAACTCCT [NM000418] +1902 g CAgTCCTCTggCCAgAgAg 5.1 1221-1239 CACCgCATgTACAAACTCCC (52) Table 2 Allele frequencies for IL-4 polymorphism Graves' disease Autoimmune All patients Controls (n=138; 276 hypothyroidism (n=215; 430 (n=101; 202 chromosomes) (n=77; 154 chromosomes) chromosomes) chromosomes) IL-4 -590 c allele 259 (0.94) 143 (0.93) 402 (0.93) 173 (0.86) IL-4 -590 t allele 17 (0.06)** 11 (0.07)* 28 (0.07)*** 29 (0.14) *P « - cι =0.006 vs controls, Pc=0.07 * * P«. ct =0.00003 vs controls, Pc=0.0004 * * *P « . « O.00001 vs controls, Pc<0.00001 Table 3 Genotype frequencies for IL-4 polymorphism Graves' disease Autoimmune All patients Controls (n=I38) hypothyroidism (n=215) (n=10I) (π=77) IL-4 -590 c/c 122 (88%) 68 (88%) 190 (88%) 75 (74%) IL-4 -590 c A 15 (12%)** 7 (9%)* 22 (11%)*** 23 (23%) IL-4 -590 l/t !(<!%) 2 (3%) 3 (1%) 3 (3%) *P=0.0I vs controls, X2=6.6, OR 0.3 (0.1-O.8, Pc=0.I I **P=0.005 vs controls, X 2 =8, OR 0.4 (0.2-0.7), Pc=0.06 ***P=0.007 vs controls, X 2 =l 1.4, OR 0.4 (0.2-0.7), Pc=0.008

Claims: Claims : 1. A method of screening a human subject for predisposition to autoimmune thyroid disease, which method comprises determining the genotype of the human subject at the -590 position of the promoter region of the 11-4 gene. 2. A method as claimed in claim 1 wherein the process of determining the genotype of the human subject at the -590 position of the promoter region of the 11-4 gene is carried out using PCR analysis. 3. A method as claimed in claim 2 wherein the PCR analysis is performed by: (a) • performing a PCR .reaction on genomic DNA isolated from the said human subject using the primer pair 5' -CTAAACTTgggAgAACATTgTC and 5'- CACTTggggCCAATCAgCA; (b) performing a PCR reaction on genomic DNA isolated from the said human subject using the primer pair ' 5'-CTAAACTTgggAgAACATTgTT and 5'- CACTTggggCCAATCAgCA; wherein the presence of an amplification product in PCR reaction (a) indicates the presence of at least one 11-4 -590c allele in the genome of said subject and the presence of an amplification product in PCR reaction (b) indicates the presence of at least one 11-4 -590t allele in the genome of said subject. 4. A method of screening a human subject for predisposition to autoimmune thyroid disease, which method comprises determining. the genotype of the human subject at one or more polymorphic loci in linkage disequilibrium with the IL-4 -590 c/t polymorphism. 5. A method of establishing the or any genetic basis for autoimmune thyroid disease the symptoms of which are manifested in a human subject, which method comprises determining the genotype of said human subject at the -590 position of the promoter region of the IL-4 gene or at one or more polymorphic loci in linkage disequilibrium with the IL-4 -590 c/t polymorphism.

Inventors: BELL JOHN IRVING, HUNT PENELOPE JANE, MARSHALL SARA ELIZABETH FARAH, BELL, JOHN, IRVING, HUNT, PENELOPE, JANE, MARSHALL, SARA, ELIZABETH, FARAH
Applicants: BELL JOHN IRVING, HUNT PENELOPE JANE, ISIS INNOVATION, MARSHALL SARA ELIZABETH FARAH, BELL, JOHN, IRVING, HUNT, PENELOPE, JANE, ISIS INNOVATION LIMITED, MARSHALL, SARA, ELIZABETH, FARAH
Application year: 2001
Main classification: C12Q1
IPC codes: C12Q1