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ClinicalScience (1997) 93,479-491 (Printed in Great Britain) 479 Editorial Review Genetic susceptibility to the development of autoimmune disease Joanne HEWARD and Stephen C. L. GOUGH* University of Birmingham, Edgbaston, Birmingham B /52 T H , U.K., and *Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 555, U.K. 1. Autoimmune diseases are common conditions which appear to develop in genetically susceptible individuals, with expression of disease being modified by permissive and protective environments. Familial clustering and data from twin studies provided the impetus for the search for putative loci. Both the candidate gene approach in populationbased case-control studies and entire genome screening in families have helped identify susceptibility genes in a number of autoimmune diseases. 2. After the first genome screen in type 1 (insulindependent) diabetes mellitus it seems likely that most autoimmune diseases are polygenic with no single gene being either necessary o r sufficient for disease development. Of the organ-specific autoimmune diseases, genome screens have now been completed in insulin-dependent diabetes mellitus and multiple sclerosis. Furthermore, the clustering of autoimmune diseases within the same individuals suggests that the same genes may be involved in the different diseases. This is supported by data showing that both HLA (human leucocyte antigen) and CTLA-4 (cytotoxic T-lymphocyte-associated-4)appear to be involved in the development of insulindependent diabetes mellitus and Graves’ disease. 3. Genome screens have also been completed in some of the non-organ-specific autoimmune diseases including rheumatoid arthritis, inflammatory bowel disease and psoriasis. Many candidate genes have also been investigated although these are predominantly in population-based case-control studies. 4. Substantial progress has been made in recent years towards the identification of susceptibility loci in autoimmune diseases. The inconsistencies seen between case-control studies may largely be due to genetic mismatching between cases and controls in small datasets. Family-based association studies are being increasingly used to confirm genetic linkages and help with fine mapping strategies. It will, however, require a combination of biology and genetics, as has been necessary with the major histocompatibility complex in insulin-dependent diabetes mellitus, to identify primary aetiological mutations. INTRODUCTION Autoimmune diseases occur when an immune response is directed against a specific organ or a number of organs and systems within an individual. Failure in immune tolerance, where tissue is recognized as ‘foreign’ instead of ‘self leads to disturbed function and often failure of a specific organ or organs. Autoimmune diseases can be separated into two groups - those that are ‘organ-specific’ and those that are ‘non-organ-specific’. Organ-specific autoimmune diseases include, for example, type 1 (insulin-dependent) diabetes mellitus, Graves’ disease, Hashimoto’s thyroiditis and multiple sclerosis. Non-organ-specific diseases include systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, psoriasis and inflammatory bowel disease. Autoimmune diseases are common within the general population and although the causes are largely unknown, it appears that development of disease in genetically susceptible individuals can be modified in permissive and protective environments. Evidence for the role of genetic factors in these diseases has been provided by looking at disease clustering within families and concordance rates in both monozygotic and dizygotic twins. As monozygotic twins share an identical genetic makeup, the higher concordance rates in these twins compared with dizygotic twins (see individual diseases for details) are to be expected if genetic components are involved in the disease process. However, concordance rates for the various autoimmune diseases Key words: autoimmunity, cytotoxic T-lymphocyte-associated4 gene, human leucocyte antigen. inflammatory bowel disease, insulin-dependent diabetes mellitus, juvenile chronic arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, thyroid disease. Abbreviations: CTM-4 gene, cytotoxic T-lymphocyte-associated4 gene; HlA, human l e u c q t e antigen; hsp, heat-shock protein; IDDM, insulin-dependent diabetes mellitus; IL-Ira, interleukin- I receptor antagonist: JCA,juvenile chronic arthritis; LMP, large multifunctional protease; MBP, myelin basic protein; MHC, major histocompatibility complex; MS, multiple sclerosis; SLE, systemic lupus erythematosus; TNF, tumour necrosis factor; TSHR. thyroid-stimulating hormone receptor; VNTR, Mn’able number of tandem repeats. Correspondence:D r S. C. L. Cough. 480 J. Heward and S. C.L. Gough are well below 100% suggesting that susceptibility to disease is not only due to genetic components but that other factors, such as the environment, play a role. The multifactorial nature of autoimmune diseases makes the study of causative factors problematic. After pioneering work in insulin-dependent (type 1) diabetes mellitus it seems likely that most autoimmune diseases are ‘polygenic’ implying the involvement of many genes [l].As no single gene has been shown to be either necessary for the development of the disease or sufficient to cause it, common disease genes are, therefore, referred to as encoding susceptibility. The involvement of more than one susceptibility gene in the disease process means that the effect of a single gene is likely to be small. This concept highlights the greatest difficulty in attempting to ‘hunt-out’ susceptibility disease genes, namely establishing sufficiently sized datasets. There are two approaches in humans which have been used to try and identify susceptibility genes of common diseases: first the candidate gene approach and second entire genome screening. A number of candidate genes have been hypothesized and tested, predominantly in population-based case-control studies. Case-control studies attempt to show the association of a gene to disease in a ‘disease-free’ population compared with a ‘diseased’ population. As we shall see later these have yielded contrasting results when the same candidate has been tested in different datasets. Such studies requiring hundreds of patients and controls have, in practice, often been performed on too few subjects. It is highly likely, therefore, that non-genetic false positives or association artefacts have arisen because of random variation as the result of a chance event. It is also impossible to eliminate population stratification [2] from such studies and it remains unclear as to which individuals are most suitable to act as a control group. An alternative to the population-based casecontrol study is the family-based study which eliminates many of the problems mentioned above. The testing of transmission of alleles of candidate genes from parents to affected and unaffected siblings using intrafamilial association tests such as the transmission disequilibrium test are tests not only of allelic association but also genetic linkage [3]. Based on the laws of simple Mendelian inheritance, the transmission disequilibrium test assumes that a heterozygous parent for a genetic variant will transmit each allele with equal frequency to any offspring. A statistically significant excess of transmission of the candidate allele to a group of offspring with disease provides evidence of allelic association. In order to detect single-gene effects, large numbers of families with parents are needed. While the family approach is more robust, collection of familial DNA samples is much more difficult than simply collecting patients and controls as many of the autoimmune diseases do not manifest themselves until the patient is middle aged, by which time one or both parents may be deceased. The collection process is also more time-consuming involving home visits to parents and relatives often at weekends in out of work hours. However, this type of study is particularly important in revealing transmission of disease genes through families and is a sensitive method for detecting susceptibility genes in polygenic diseases [4-61. The second approach of entire genome screening and linkage analysis makes no assumption of the gene or genes involved in the development of the disease, disease gene frequencies or mode of inheritance of genes. Studies already performed in type 1 diabetes [7, 81, multiple sclerosis [9-111, psoriasis [12, 131 and inflammatory bowel disease [14] have made use of genetic markers (microsatellites) from existing genetic maps. The markers have been analysed in datasets of large numbers of families in which DNA is available from two affected siblings with or without parental DNA. Genetic linkage of a marker to disease is said to be present when there is a significant excess of alleles shared identical by descent, when parental DNA is available for analysis, and identical by state when parental DNA is not available. Results from the completed genome searches have found regions of linkage, some of which reassuringly coincide with previous candidate loci including human leucocyte antigen (HLA). They have also confirmed that the common multifactorial diseases are polygenic in nature. Clinicians have long since noted that it is not uncommon to diagnose more than one autoimmune disease in a single patient; for example, autoimmune thyroid disease is frequently seen in patients with type 1 diabetes. Autoimmune diseases also seem to cluster within families. This has been confirmed by the 5th Genetic Analysis Workshop [15] with the finding of at least one case of autoimmune thyroid disease in 40% of type 1 diabetic families, a result supported by data from the British Diabetic Association Warren Repository where 22% of type 1 diabetic families had at least one case of autoimmune thyroid disease (S. C. Bain, personal communication). These results imply that the same genes may be involved in different autoimmune diseases. The remainder of this review presents an overview of results obtained to date. ORGAN-SPECIFIC AUTOIMMUNE DISEASES Insulin-dependent diabetes rnellitus Insulin-dependent diabetes mellitus (IDDM) is characterized by T-cell-mediated autoimmune destruction of the pancreatic /?-cells leading to a deficiency in insulin production resulting in a lifelong requirement of insulin replacement by injection. The disease may also lead to the development of complications including eye disease (diabetic retinopathy), renal disease (diabetic nephropathy), 48 I Genetic susceptibility to autoimmune disease nerve damage (neuropathy) and vascular disease (including peripheral vascular disease, coronary artery disease and cerebrovascular disease). It is a common childhood condition affecting around 1 in 300-400 children with a concordance rate in monozygotic twins of 36% [16, 171. Genetic susceptibility to type 1 diabetes has been established from candidate gene and whole genome screen studies and has recently been reviewed [l, 181. The major findings only, therefore, will be summarized here. makes it extremely difficult to distinguish the ‘true’ susceptibility allele from alleles in linkage disequilibrium. For example, in Caucasians DR3 and DR4 along with associated DQ alleles are increased in patients with diabetes but it has been almost impossible to define which of these alleles is responsible for association of the HLA region with disease. In order to overcome difficulties due to linkage disequilibrium, studies of many different ethnic groups have been performed using the hypothesis that the ‘true’ susceptibility allele of the MHC class I1 region will be present in all races. Results from studies combining both genetic and biological approaches have shown conclusively that the HLA DQA1, DQBl and DRBl encode primary aetiological determinants in the MHC diabetic locus. However, in some ethnic groups, additional susceptibility loci must lie outside the class I1 region because DR3, for example, can be stratified by class I polymorphisms [191. Genuine differences in associations between different genes in the MHC and type 1 diabetes may be seen in various ethnic groups because of differences in allele frequencies at the susceptibility locus and at other interacting loci that may be unlinked. Cross-ethnic group comparisons are therefore only informative when results are positive. A negative result in one ethnic group does not mean that positive results in other ethnic groups are false. Other genes within the MHC that have been suggested as candidate genes for IDDM determinants include the transporter associated with antigen processing (TAP) genes [20, 211 and the large multifunctional protease (LMP)genes [22]. TAP is a Major histocompatibility complex (MHC) class II region Major histocompatibility complex (MHC) class I1 molecules are assembled in the endoplasmic reticulum and are transported through the Golgi and trans-Golgi reticulum to the cytosol where they take up degraded antigen, and are transported to the cell surface to present the antigen to CD4+ T-cells. These T-cells attack foreign antigen presented by the MHC class I1 molecules either directly or by activating cells such as macrophages. This interaction between MHC class I1 molecules and T-cells makes the MHC class I1 region a strong candidate region for diseases which develop as a result of T-cell-mediated autoimmunity. The HLA class I1 region of the MHC on chromosome 6p21 (IDDMI) (Fig. 1) accounts for 35% of familial clustering in IDDM and is by far the major susceptibility locus [l]. Many studies have been performed on the DRB1, DQAl and DQBl regions which are in strong linkage disequilibrium [19]. This Class Il region TAP DP DN R B2A2BlAl A 3 DM LMP LMP 9 2 A2 9 3 91 Al 21921 B AB DR DQ RTAP DO 91 92 9 3 99 A I000 Class I11 region c4 Bf TNF HSP7O C2 2 1 2000 A B Class I region B C X E J A H G F Fig. 1. MHC class I, II and 111 genes. TAP, transporter associated with antigen processing; LMP, large multifunctional protease; HSP70, heat-shock protein 70; TNF, tumour necrosis factor. 402 J. Heward and S. C.L. Gough heterodimer composed of TAP1 and TAP2 which translocates degraded antigens into the endoplasmic reticulum for antigen processing by MHC class I molecules. TAP is polymorphic and situated adjacent to the HLA-DQ locus. Results from many different races including Sardinians, Finnish, Norwegians and Japanese have shown no primary association between TAP and IDDM and the differences in frequency of the TAP alleles appear to be the result of linkage disequilibrium with DQ [23, 241. LMP2 and LMP7 are subunits of a subset of proteasomes which are large molecular assemblies with multiproteolytic activities believed to degrade damaged and unwanted cellular proteins. Recent work by Deng et al. [22] indicates a role for LMP7 in IDDM with an increase in allele A being observed in both case-control and family studies. The authors claim an association independent of linkage disequilibrium with the D R D Q region. Subsequent work by Undlien et al. [25] has, however, shown no independent association of either LMP2 or LMP7 polymorphisms with susceptibility to IDDM. The discrepancy in results between these two studies appear to be due to the failure of Deng et al. [22] to include the information from DR4 subtyping. A combination of biology and genetics has been necessary to determine primary aetiological determinants in the MHC. Such an approach, which has also been adopted for the insulin gene locus, will undoubtedly be necessary at other loci to pinpoint aetiological variants. unknown, insulin gene expression appears to be influenced by the VNTR genotype [26]. insulin gene region Other genes The insulin gene (INS) region (IDDMZ) on chromosome llp15.5 contributes around 10% of the familial clustering seen in type 1 diabetes [l]. Having originally been postulated as a candidate gene for type 2 (non-insulin-dependent) diabetes, allelic variation at the INS VNTR (variable number of tandem repeats) was first shown to be associated with type 1 diabetes in case-control studies and later linked in family-based studies (for review see [26]). IDDM2 has been mapped to a 4.1 kb region surrounding INS [27] and after the identification of 13 common polymorphic sites, the susceptibility locus has been shown to lie within the VNTR [5]. There are three classes of VNTR, classes I, I1 and I11 averaging 570, 1200 and 2200 bp respectively. Although susceptibility to type 1 diabetes may be conferred by the shorter class I VNTR alleles, population studies show that class I11 VNTR alleles are dominantly protective and are associated with a 60-70% reduction in the risk of developing the disease [26]. Further interest in this region relates to an observation that transmission of predisposing INS VNTR alleles to type 1 diabetic offspring is related to parent of origin [26]. Although the mechanism linking variation at the INS VNTR and type 1 diabetes remains Recent genome-wide searches have identified 14 further putative loci as contributing to type 1 diabetes [l, 181. Additional family studies are under way to confirm these loci, and fine mapping strategies are ongoing to ultimately identify specific mutations causing disease. Table 1 lists the current replicated IDDM loci. Cytotoxic T-lymphocyte-associated-4 gene As previously mentioned, certain susceptibility genes may be common to more than one autoimmune disease. This could explain the clustering of different autoimmune diseases within families and the same individuals. The cytotoxic T-lymphocyteassociated4 (CTLA-4) gene on chromosome 2q33 was first identified as a candidate gene in Graves’ disease [28] but is an equally strong candidate for other T-cell-mediated autoimmune diseases. Recent studies in vifro and in v i m have shown that CTLA-4 may downregulate T-cell function (for review see [29]). Further work has shown that CTLA-4 encodes a T-cell receptor that may inhibit CD28-dependent interleukin-2 production [30]. A recent study by Nistic0 et al. [31], examining an A to G polymorphism in the leader peptide of exon 1 of the CTLA-4 gene, showed an increased frequency of the G allele in a Belgian case-control study and increased transmission of the G allele to diabetic offspring in Italian and Spanish family studies. Failure to replicate in additional family sets from the U.K., U.S.A. and Sardinia is most likely to be the result of heterogeneity between the different ethnic groups studied. Increasing evidence in Graves’ disease, however, suggests that CTLA-4 is a susceptibility locus for autoimmune disease and therefore may well play a role in type 1 diabetes. Graves’ disease Graves’ disease is an autoimmune disease of the thyroid gland which is characterized by an overactive thyroid gland (hyperthyroidism), a diffuse goitre and in some cases ophthalmopathy and pretibial myxoedema. The frequency and severity of the symptoms, however, varies between individuals. Overactivity of the gland is the direct result of the stimulating effect of an autoantibody directed against the thyroidstimulating hormone receptor (TSHR) [32]. The disease usually presents in the fourth decade of life and is reported to be 7-10 times more common in women than men. The reason for the female preponderance is unknown but as with other organspecific autoimmune diseases a number of hypotheses Genetic susceptibility to autoimmune disease Table 1. IDDM susceptibility loci. INS, insulin gene; GCK, glucokinase gene. See text for references. Locus name Chromosome location IDDMI [HM) IDDM2 [INS) IDDM3 IDDM4 6p2 I I lp21 lDDM5 IDDM6 IDDM7 IDDMB IDDM9 IDDMI0 IDDMI I CTIA-4 [IDDMIZ) IDDMI3 GCK 1% I lq13 $25 18q 2q3 I 6q27 3q21-q25 lop1 1.2-ql1.2 14q24.3-q3 I 2q33 2q34 7P have been put forward. First, susceptibility loci may be located on the sex chromosomes. Currently there is no published evidence to substantiate this. Second, females may have an increased immune responsiveness. Third, observed differences may be related to differences in sex hormones. Testosterone suppresses and oestrogens exacerbate experimental autoimmune thyroiditis. Therefore, a stronger genetic influence may be required to overcome the inhibitory effects of testosterone for Graves’ disease to develop in males. As with type 1 diabetes, evidence for the existence of genetic susceptibility associated with permissive and protective environments is supported by epidemiological data [33]. Although most cases of Graves’ disease occur in unrelated individuals (sporadic), 10-20% appear to cluster within families. The concordance rate in identical (monozygotic) twins appears to be in excess of 30%, but falls well short of 100%. The concordance rate in non-identical twins is around 3-9%. The dissection of susceptibility genes in Graves’ disease is not as advanced as that seen in type 1 diabetes and most reports have concentrated on candidate genes in populationbased case-control studies. MHC class II region The target of the autoimmune process in Graves’ disease is the follicular cell. This, and activated lymphocytes in patients with Graves’ disease exhibit aberrant expression of MHC class I1 antigens including the DR antigen. The MHC HLA region on chromosome 6 is therefore an obvious candidate for Graves’ disease. Almost all studies examining the relationship between the HLA region on chromosome 6 and autoimmune thyroid disease are population-based case-control studies. Most have found associations with specific alleles of the class I1 region. It is likely that inconsistent results from early studies and those 483 from more recent reports describing independent associations within the class I1 region, differences between male and female populations and age at onset of disease have arisen because of datasets with too few subjects. While some of the reported differences between populations from different races and geographical locations may also be the result of a chance finding in a small dataset, it is highly likely that differences do exist [34, 351, as has been seen in the larger family studies in type 1 diabetes [l]. There is increasing evidence supporting an association between Graves’ disease and HLA-DR3 at least in Caucasian populations. However, DR3 is in strong linkage disequilibrium with DQB1*0201 and DQA1*0501, both of which are strongly associated with Graves’ disease [36, 371. As with type 1 diabetes it will be extremely difficult to determine the primary susceptibility locus. Studies implicating independent HLA associations and differences between males and females need to be repeated with larger numbers of subjects before meaningful conclusions can be drawn. There are very few family-based studies available for review [38-401. Those that have been reported have failed to replicate, with linkage analysis, results obtained in population-based case-control studies [38]. The reason for this is almost certainly the result of too few families within the datasets with studies lacking the power to detect linkage. Moreover, the largest reported study to date also included families of different ethnic backgrounds [38]. However, a combined segregation and linkage analysis of patients with Graves’ disease and associated autoantibody status showed linkage to HLA-DR [39]. A more recent family-based study implicates a role for DPBl in distinguishing autoantibody-positive family members who develop Graves’ disease (DR17-DQ2-DPB1*0101) from those who would remain euthyroid (DR17-DQ2-DPB1*0401) [40]. As approximately 10% of the general population have thyroid autoantibodies, but not clinical thyroid dysfunction, replication of the findings of Ratanachaiyavong et al. [40] in further datasets is needed to determine whether HLA typing will help identify autoantibody-positive individuals likely to develop thyroid disease. CTLA-4 gene Yanagawa et al, [28] looked at exon 3 of the C T U - 4 gene region which contains an (AT)n repeat in the 3’ untranslated region and observed 21 alleles ranging in size from 88 to 134 bp. A significant excess of allele 106 was observed in patients with Graves’ disease in a case-control study. The result was most evident in females with protective HLA haplotypes (DQA1*0201 positive/DQA1*0501 negative). Although the number of AT repeats may be important for mRNA stability, the disease sus- 484 J. Heward and S. C.L. Gough ceptibility mutation within CTLA-4 gene remains unknown. As previously mentioned, Nistico et al. [31] examined an A to G polymorphism in exon 1 (which is in linkage disequilibrium with allele 106 in exon 3) of CTLA-4 and found association of the G allele with IDDM. This result was replicated in a casecontrol study of Hong Kong Chinese patients with Graves’ disease [31]. Similar associations have since been reported in Caucasian Graves’ subjects from Germany, Canada [41] and the U.K. [42], indicating that this may be a common gene to both type 1 diabetes and Graves’ disease. Thyrotropin (thyroid-stimulating hormone) receptor gene The fact that Graves’ disease develops as a result of antibodies stimulating the TSHR [32], makes the TSHR gene an obvious candidate for the disease. Polymorphism at the first position of codon 52 (C52-A52) changing a proline into a threonine has been described [43]. Although early case-control studies report association of this polymorphism with disease [44], this result has not been replicated in other datasets [45]. Combined segregation and linkage analysis using three microsatellite markers within the TSHR gene introns in a family-based study failed to provide evidence for genetic linkage to Graves’ disease [46]. Although this represents one of the largest family studies in Graves’ disease to date, too few affected sib-pairs were available to completely exclude an effect on disease susceptibility. From our knowledge of type 1 diabetes it is likely that Graves’ disease is a polygenic disorder in which each gene contributes between 5 and 10% to genetic susceptibility. It is unlikely, therefore, that datasets the size of that used by De Roux et al. [46] will be of sufficient size to exclude most genetic determinants to Graves’ disease. Tomer et al. [47] recently reported linkage analysis of eight candidate gene regions including the TSHR gene on chromosome 14q31. This study looked at 109 family members from 19 Caucasian North American and Italian families, which included only 14 patients with Graves’ disease and 32 with Hashimoto’s thyroiditis. The microsatellite Dl4S8l gave the highest positive LOD score, although this marker is a considerable distance from the TSHR gene (approximately 25 centiMorgans). This result, which needs replicating, places a Graves’ susceptibility gene (GD-1) in a similar region to ZDDMll, but it is unlikely that either of these loci correspond to the TSHR, and therefore it is unlikely that the TSHR is the gene in this region responsible for conferring primary susceptibility to disease. Interleukin- I receptor antagonist gene Interleukin-1 (IL-1) can modify the functions of thyroid cells in v i m . It is produced in the mono- nuclear infiltrate by thyroid cells and there is evidence that IL-1 produces fibroblast activation in thyroid-associated ophthalmopathy. The interleukin1 receptor antagonist (IL-lra) is a 22-25kDa protein that is related to IL-lcr and IL-1p. Although IL-lra competes with IL-lcr and p to occupy cell-surface receptors, it does not stimulate signal transduction thus inhibiting IL-1 action. A VNTR in intron 2 of the ZL-lru gene (ILlRN) gives rise to five alleles [48]. The two common alleles, ILlRN*l (which has four copies of an 86 bp repeat) and ILlRN*2 (which has two copies of the same repeat) account for 95% variability at this locus. ILlRN*2 is a marker of chronic inflammatory disease and is increased in patients with Graves’ disease [48]. This could be due to linkage disequilibrium with other genes on chromosome 2 or it could be functionally significant as each 86 bp repeat has possible transcription factor binding sites. This association has only been detected in a case-control study. Until the association of ILlRN*2 is shown in family studies, population stratification and genetic mismatching between cases and controls cannot be excluded as a likely explanation. No association has been found between ILlRN*2 and thyroid antibody levels, or other clinical features of Graves’ disease. Hashimoto’s thyroiditis Hashimoto’s thyroiditis is characterized by hypothyroidism, a diffuse goitre and the presence of autoantibodies to thyroglobulin and thyroid peroxidase. Few genetic studies have been performed and those that have generally look at the HLA region. Again, conflicting results have been obtained [38, 49-51]. The first studies performed in Caucasians showed non-significant increases in DR3 and DQw2 and no association with DPB in a case-control study [49]. These results were not replicated in a subsequent case-control study that found no association with DRBl or DQBl but an association with DQA1*0402 [50]. The same authors also performed a meta-analysis of several previous studies and demonstrated weak, positive associations between disease and DR3 and DR4, suggesting that their lack of association with DRBl and DQBl could be due to sample size. A family-based study found DR5 (DRll+DR12) to be increased in family members but all LOD scores were negative leading the authors to suggest that it is unlikely that Hashimoto’s thyroiditis is linked to the HLA region in Caucasians [38]. However, as previously mentioned, the numbers of subjects in this study were small. A Japanese case-control study showed an increase in DRB4*0101 and HLA-A2 with the presence of both alleles conferring increased risk of disease [51]. DQA1*0102 was decreased suggesting a putative protective role. Further studies need to be performed to elucidate the true role of HLA in this disease along with further genetic studies looking at the roles of other candidate genes. Genetic susceptibility to autoimmune disease In addition to showing allelic association of a microsatellite of C T U - 4 (allele 106) with Graves’ disease, association has also been reported with Hashimoto’s thyroiditis [42]. This association was reported, however, in a small case-control study and needs replicating in family-based studies. Multiple sclerosis Multiple sclerosis (MS ) is a chronic inflammatory demyelinating disease of the central nervous system that results in a number of sensory and motor neurological manifestations. The disease probably results from T-cell-mediated destruction of the myelin sheath in genetically susceptible individuals. Epidemiological evidence provides good support for a genetic basis to the development of MS. Increased familial risks range from 30% for monozygotic twins [52] to 3-4% for first-degree relatives [53]. By screening 15 000 individuals with MS, Ebers et al. [54] reported an increased frequency of disease among first-degree biological relatives. The frequency of MS among first-degree non-biological relatives (adopted relatives), however, was no greater than that of the expected population prevalence. These data imply no detectable shared environmental effect on disease aetiology. In the only study of conjugal pairs of MS the crude recurrence in children rose to 1 in 17 [%], which is significantly higher than reported population-based risks for offspring of single affected parents (1 in 200), demonstrating the importance of the inheritance of genetic susceptibility loci from both parents. MHC class II region Much attention has been paid to the roles of the genes within this region in susceptibility to MS in many different races and, as in the other diseases, reviewed results are conflicting [56, 571. Associations with DR2 have been reported in numerous Caucasian populations. Increases in DQAl*O102 and DQB1*0602have also been observed with 96% of MS patients in one study having a DQA allele with glutamine at residue 34 and a DQB allele encoding DQB chains sharing long polymorphic stretches [58]. However, no excess of DQB1*0302 and DQB1*03032 was observed in patients with MS; these alleles share hypervariable regions with DQB1*06, implying that if MS is related to particular DQB alleles it does not appear to be due to the hypervariable regions of these alleles. Further studies have placed associations to DQBl sequences and position 34 of DQA as secondary to linkage disequilibrium with the haplotype DRB1*1501-DQA1*0102-DQB1*0602 due to the absence of over-representation of the DQA/DQB heterodimers in DR15-negative patients [591* 405 Myelin basic protein The myelin basic protein (MBP) gene is on chromosome 18 and consists of seven exons. A study looking at two tetranucleotide repeats 5‘ to exon 1 of the MBP gene in Shanghai Chinese revealed that no allele was associated with MS [60]. However when the (TGGA), polymorphism 5’ to the MBP was studied in Danish patients with MS, three different band patterns were observed with the 450 bp band being significantly increased in patients with MS [61]. Further studies in Danish patients with MS have revealed other polymorphisms in this region which were also associated with MS [62]. These data from case-control studies suggest that polymorphisms of the MBP gene may play a role in MS but further studies in families are required for confirmation. Other susceptibility loci Several genome screens have recently been completed in families with MS. Sawcer et al. [9] found linkage at chromosome 17q22 and 6p21 (MHC) along with several other regions using affected sibpairs, but could not replicate susceptibility loci in extra datasets. The Multiple Sclerosis Genetics Group using affected sib-pairs and affected relative pairs identified 19 regions that may be important in MS including the MHC but no locus generated overwhelming evidence of linkage [lo]. Finally, Ebers et al. [ l l ] identified five loci on chromosomes 2,3,5,11 and X but found no evidence of linkage in the HLA region. However, a marker just outside the HLA region showed significant evidence for linkage disequilibrium in all datasets studied. These studies, although providing candidate regions which can be followed up in future genetic studies, highlight the difficulties faced by geneticists when attempting to replicate the findings of a polygenic trait in different populations. Other diseases Other organ-specific autoimmune diseases include myasthenia gravis and Addison’s disease. The autoimmune process in myasthenia gravis results in postsynaptic blockade of neuromuscular conduction by autoantibodies directed against the acetylcholine receptor. There is a 40% concordance rate between monozygotic twins and an increased incidence of disease in relatives of patients with myasthenia gravis [63]. An increased frequency of autoimmune thyroid disease has been reported in families of patients with myasthenia gravis providing further evidence for clustering of autoimmune diseases [64]. Candidate genes for myasthenia include the MHC [65], immunoglobulin genes [66], T-cell antigen 406 J.Heward and S. C.L. Gough receptor genes [67] and the acetylcholine receptor gene. The role of the gene encoding the CI subunit of the acetylcholine receptor has been investigated [68]. Two polymorphic sites, H B and BB, within the first intron have been identified. In a family-based study, the HB*14 allele was consistently transmitted to the affected offspring from the parent implicating a dominant role for the allele in disease susceptibility [68]. Importantly, this result has been replicated by Heckmann et al. [69]. Addison’s disease results from the autoimmune destruction of the adrenal glands leading to dysfunction of steroidogenesis. The disease can develop independently or as part of autoimmune polyglandular syndrome type I1 in which it occurs with other autoimmune diseases including IDDM and autoimmune thyroid disease. Associations have been found with HLA-B8, DRB1*0301, DQA1*0501 and DQB1*0201 [70-721. The major autoantigen in Addison’s disease, steroid 21-hydroxylase, has a functional gene (CYP21B) and a pseudogene ( C W 2 I A ) located in the HLA class I11 region on chromosome 6 [73]. A primary role for polymorphic sites within these genes is difficult to ascertain as a result of strong linkage disequilibrium with other genes within the HLA region including class I1 loci. NON-ORGAN-SPECIFICAUTOIMMUNE DISEASES Systemic lupus erythematosus Systemic lupus erythematosus (SLE) is characterized by many abnormal immune reactions and clinical symptoms. Autoantibodies are produced against double-stranded DNA, intracellular ribonucleoproteins, haematological cells and phospholipids. Autoantibody profiles differ among patients but the same pattern is consistent in individuals. Some of the autoantibodies produced are directly pathogenic, for example, anti-erythrocyte, antiphospholipid and antiplatelet antibodies, while the antinuclear antibodies can cause disease due to the tissue damage that results from the immune system trying to neutralize their actions. SLE can occur at any age but the second to the fifth decade of life is the most common time for the disease to manifest itself. Although 10-12% of patients have first-degree relatives with SLE [74] affected family members usually present at a similar time point rather than age suggesting the involvement of external environmental triggers [75]. Although a number of environmental stimuli have been postulated including sex hormones, UV light, viral infections and diet, genetic susceptibility is highly likely with 1.7-3% of firstdegree relatives of patients developing SLE compared with 0.2-0.3% in the general population [74, 751. Concordance rates also support the involvement of genetic factors in SLE with a rate of between 24 and 69% in monozygotic twins compared with 2-9% in dizygotic twins [76]. MHC class II region Early reports looking at the involvement of HLA in SLE linked DR2 and DR3 to disease but later reports indicated that these alleles have an increased association with the production of autoantibodies rather than with the disease itself [75]. The production of antiRo (Ro is a small nuclear and cytoplasmic ribonucleoprotein of unknown function) and antiLa (La is a transcriptional termination factor for RNA polymerase) correlate with the presence of DR2, DR3, DQ1 (DQ5 and DQ6) and DQ2 [77, 781. Autoantibodies to Ro are found in 25-50% of patients with SLE and autoantibodies to La usually accompany them. The highest levels of antiRo and antiLa antibodies are found in patients who are heterozygous for D Q 5 D Q 6 and DQ2 suggesting that the production of these autoantibodies is more dependent on D Q than D R [77, 791. Other autoantibodies are present in patients with SLE but the HLA associations with these are less clear. Antiphospholipid antibodies are found in 34-44% of SLE patients and an increased frequency of DR7DR4-positive patients carry anticardiolipin antibodies [80]. These are in linkage disequilibrium with DRB4 suggesting that this may be the primary association. Studies of different races have identified a-myriad of HLA associations with SLE: DQAl* 0501 (in linkage disequilibrium with DR3) being strongly associated in Scandinavians [81], DR15 (subtype of DR2) in Southern Chinese [82] and DR3 and DR2 in Germans [83]. These results indicate that both DR3 and DR2 (primarily 1501 and 1601) and their associated D Q alleles play a role in SLE. Increases have also been observed in DPB1*0101 but again this is in linkage disequilibrium with DR3. Unfortunately all results to date have been obtained from population-based casecontrol studies and need to be confirmed in familybased studies. Heat-shock protein genes Heat-shock proteins (hsp) are highly conserved proteins synthesized after stressful stimuli. The expression of hsp90 is found to be increased in the mononuclear cells of about 25% of patients with SLE and antibodies to this protein are detected in patients with SLE. Those patients with increased antibody production are more likely to have renal disease and low C3. Another heat-shock protein thought to play a role in SLE is hsp70. The hsp70-1, hsp70-2 and hsp-horn genes produce products with highly similar sequences but they differ in their regulation. Hsp70-2 encodes a protein functionally relevant to antigen processing and has been implicated in autoimmune disease in Caucasians. Associ- Genetic susceptibility to autoimmune disease ation of a polymorphism (A to G transition) in the coding region of the hsp70-2 gene with SLE in African Americans independent of DR3 or the C4A deletion has been reported in a case-control study [84]. This awaits confirmation in families. Tumour necrosis factor gene Tumour necrosis factor (TNF) is an inducible cytokine with a broa; range of actions including increased HLA class I and I1 expression and increased B- and T-cell proliferation. Macrophages are the major source of TNF but it is also present in other cells such as skin cells and B- and T-cells. A polymorphism (guanosine to adenosine substitution) in the promoter region, giving rise to a rare TNF gene allele, has been found to be increased in patients with SLE in a case-control study, but is probably due to linkage disequilibrium with DR3 [85]. This is supported by the observation that TNF is more strongly associated with autoantibody production than with the disease itself but only when in association with DR3. This suggests that the TNFa polymorphism, TNF2, plays a role in susceptibility to disease on a DR3 haplotype but not independently. Rheumatoid arthritis Rheumatoid arthritis was originally described as a chronic inflammatory disease of peripheral joints but is now recognized as a chronic or subacute systemic inflammatory disorder. Concordance rates in monozygotic twins vary between 12 and 30% and a 2-3-fold excess of disease in females has been reported. The HLA region has been linked to disease and is thought to account for half of familial rheumatoid arthritis and a fifth of rheumatoid arthritis in the population [86]. HLA-DRB1*04 is associated with disease in Caucasians with the DRBl*O401/0404 genotype carrying a higher risk of development of more severe forms of the disease especially in young men [87]. A more recent study [88] found that DRB1*04 or DRB1*01/04 was only related to disease in seropositive patients and that rheumatoid factor was a better predictor of disease severity than HLA subtype, suggesting that the effect of these alleles on severity of disease may be linked to seropositivity. The positive association with disease of DRB1*04 and DRBl*Ol has been replicated but once more no relation between these alleles and severity of disease was noted [89]. Again small numbers in some of these studies may explain differences in results. Reported associations between a polymorphism of TNF, T-cell receptor a and loci are probably all secondary to the role played by HLA-DRB1*04 [go]. Recently rheumatoid arthritis has been linked to the N M P l gene (a macrophage resistance gene) on chromosome 2q35 487 [91] and a genome screen has confirmed linkage of rheumatoid arthritis to HLA [92]. Inflammatory bowel disease The chronic inflammatory bowel diseases including Crohn’s disease and ulcerative colitis have unknown aetiology but evidence suggests that genetic factors play a role in predisposition to these diseases. Although these diseases predominantly involve the bowel, other systems are also affected, including liver, joints and skin. The HLA region has been implicated in ulcerative colitis with an affected sib-pair study providing evidence for linkage with the HLA-DRB1 region [93]. The same study showed association of HLA-DRB1*0103 and DRB1*12 with ulcerative colitis but no association of Crohn’s disease with any HLA genes. These findings also indicated that the HLA-DR3-DQ2 haplotype predicts extensive ulcerative colitis, which is at odds with earlier work indicating that this haplotype has a protective role in the disease [94]. A recent genome screen [14], however, has identified a susceptibility locus for Crohn’s disease on chromosome 16. This result has since been replicated in a further dataset [951. Psoriasis Psoriasis is an inflammatory skin disease characterized by red, scaly skin patches usually on the scalp, elbow and knees. Different types of arthropathy are also seen in patients with psoriasis which at times can be virtually indistinguishable from rheumatoid disease. The usual age of onset is 15-30 years of age and it affects 2% of the population. The disease has a large genetic component with concordance rates in monozygotic twins of 65-70% compared with 15-20% in dizygotic twins. The risk to first-degree relatives is between 18 and 23% and inheritance of the disease fits an autosomal recessive model in these relatives [96]. Environmental factors are also thought to play a role and these include streptococcal infection and stress. MHC class I1 region The disease has been subdivided into two types; type I has an early onset and a positive family history and type I1 has a late onset and is sporadic with no family history. Type I psoriasis is associated with the Caucasian extended HLA haplotype Cw6-B57-DRB1*0701-DQA1*0201-DQB1*0303 with the class I antigens (Cw6-B57) being associated to a much higher extent than the class I1 alleles (DRB1*0701-DQA1*0201-DQB1*0303) [971, Suggesting that a gene for familial psoriasis is associated with the class I side of the extended haplotype. It is still unknown as to whether the susceptibility gene 488 J. Heward and S. C.L. Gough lies within the HLA class I genes or another gene in close linkage disequilibrium. DQA1*0501-DQB1*0301 and DRBl*08-DQAl* 0401-DQB1*0402 as susceptibility alleles and DRB1*07, DQA1*0201 and DQB1*0201 as protective alleles. Other genes A microsatellite marker on chromosome 17q was shown to be in linkage with psoriasis in a large extended white American family [12], although studies performed in extended kindreds from Northern Europe have failed to replicate this result [98, 991. A further locus on chromosome 4q has been reported in extended families from Ireland and the UK [13]. This finding awaits replication. In a recent genome-wide search using largely the 260 microsatellite markers employed in the genome screen of type 1 diabetes, four regions of preliminary linkage were identified on chromosomes 2, 8 and 20 and significant linkage was demonstrated with markers from the MHC region at 6p21 [loo]. As in type 1 diabetes this comprehensive screen indicates that a gene or genes located within the MHC region are conferring the greatest single effect on the susceptibility to disease. Juvenilechronic arthritis Juvenile chronic arthritis (JCA) is an inflammatory disease starting in children under the age of 16 years and lasting at least 3 months. There are two major types of JCA, pauciarticular and polyarticular, depending on the clinical presentation and number of joints affected. A further subgroup has a systemic onset with fever and a rash. Thirty-five percent of patients who start with pauciarticular JCA go on to develop polyarticular JCA, which leads to severe joint destruction and disability. MHC class II region The associations of JCA with the HLA region tend to vary according to the type of JCA being studied. Those with persistent pauciarticular JCA have been reported as having increased DRBl*1301, DRB1*0801, DQB1*0603,DQBl*04 and DPB1*0201 and decreased DRB1*0701 [101, 1021. Those with polyarticular JCA are reported to show an association with DRB1*0801, DQB1*04 and DPB1*0301 with a decrease in DRB1*04 [loll. A further study into seropositive and seronegative polyarticular JCA confirmed the roles of DRB1*0801, DQB1*04 and DPB1*0301 in this subgroup of the disease [103]. Studies performed on the subgroup with systemic onset JCA have implicated DRB1*04 as playing a major role in this disease. Those patients with polyarticular JCA that had a pauciarticular onset are thought to have an independent risk from DQA1*0101 [loll. Studies on the early onset pauciarticular subgroup have identified DRB1*11- Other genes Other genes implicated in JCA include the T-cellreceptor variable gene and the IL-1 gene. The T-cell-receptor gene has an M chain (TCRa) and a j? chain (TCRb). The Tcrb gene complex is on chromosome 7 and is composed of variable, diversity, joining and constant regions. The variable region (Tcrb-V) has 20 subfamilies each with between 1 and 10 members. Studies have identified a polymorphism which correlates to the Tcrb-V6.1 gene which is associated with JCA in patients with the DQA1*0101 allele [104]. Polymorphism (C to T change at position 889 ) in the promoter of IL-lci has been shown to be increased in JCA [105]. This polymorphism was increased in DPZpositive patients and decreased in DRS-positive patients. In common with many of the diseases reviewed, most of the studies reported to date in JCA are population-based case-control studies on small numbers of subjects. Although the age of presentation makes the collection of families possible, there is a considerable degree of phenotypic heterogeneity. Any genetic analysis will therefore have to take this into account. CONCLUSIONS Genetic susceptibility to the development of autoimmune disease is a complex subject with many different genes and their products interacting with each other and external stimuli. A summary of results found to date can be seen in Tables 2 and 3. Certain gene regions including HLA and probably CTLA-4 are likely to cause susceptibility to more than one autoimmune disease thereby helping to explain the clustering of diseases within the same families and individuals. Conflicting results have been obtained in many different datasets. This is partially due to the different racial and ethnic origins of the subjects but also the inadequate numbers studied and poor phenotypic characterization in heterogeneous diseases. Inconsistencies between population-based case-control studies are largely the result of genetic mismatching between cases and controls, highlighting the need for genetic studies to be carried out primarily in families. Many studies have used numbers that are too small to detect single-gene effects in polygenic diseases. This applies to both case-control and family-based studies. The combined approach of genome-wide searches in families, and targeting candidate genes in both family studies and population-based case-control Genetic susceptibility to autoimmune disease 489 Table 2. Summary of organ-specific autoimmyne disease susceptibility loci. TAP, transporter assou’ated with antigen processing; IMP, large multifunctional protease; INS, insulin gene. See text for references. ~ HlA TAP LMP INS crlA-4 IDDM1-I 0 It-Ira ~~ IDDM Graves’ disease Hashimoto’s thFoiditis Myastheniagravis Addison’s Disease MS t t t t t t t t t t t t t t t Thptropin receptor Iggenes T-cell receptor Acet$choline receptor Steroid 21-hydroxylase MBP Proteolipidprotein t t t t t t t t Table 3. Summary of non-organ-specific autoimmune disease susceptibilii lod. TAP, transporter associated with antigen pmcesing; IMP, large multifunctional protease. See text for references. SLE HLA t TAP IMP T-cell receptor Complement Heat-shock proteins TNF It-I t t t t Psoriasis Inflammatory bowel disease t t t Rheumatoid arthritis t t t t studies, has a h w e d geneticists to make substantial progress towards the identification of susceptibility loci in autoimmune diseases. The greatest advances have been made in IDDM although progress is being made in MS, inflammatory bowel disease, psoriasis and rheumatoid arthritis. Family-based association studies such as the transmission disequilibrium test are being increasingly used to confirm linkages and fine map genes in positional cloning strategies in IDDM, and may be the way forward for other autoimmune diseases. Intrafamilial association studies have the advantage over population-based case-control studies of avoiding artefactual associations due to population stratification. 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