Genetic analysis of susceptibility to type 1 diabetes.

Springer Semin Immunopathol (1992) 14:33-58

SpringerSeminars in Immunopathology ~ Springer-Verlag 1992

Genetic analysis of susceptibility to Type 1 diabetes J . A . Todd Nuffield Department of Surgery, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK

1 Introduction In 1985 three groups reported the chromosomal location of the gene responsible for cystic fibrosis (CF). Linkage analysis in families using polymorphic DNA markers limited the search to a region of DNA of about 3 x 106 base pairs (bp). It has taken 4 years to identify the CF gene on chromosome 7 [48]. It is surprising, therefore, that even though a genetic determinant of susceptibility to the autoimmune disease Type 1 diabetes was located on human chromosome 6 almost 15 years ago [65, 66, 100, 107], the genes responsible for disease development have not been identified definitively. The amount of DNA is similar: a susceptibility gene (or genes) lies within 3 X 106 - 4 • 106 bp of DNA that contains the major histocompatibility complex (MHC). The nature of the problem, however, is very different. This review describes some of the special problems that the MHC has presented for disease gene mapping, approaches designed to overcome these difficulties, strategies to help resolve current uncertainties and a view of future research into the genetic basis of Type 1 diabetes. Many of the methods and principles overlap with those applied to the CF gene search. Genetic analysis of susceptibility to Type 1 diabetes has been reviewed recently [7, 63, 103, 107, 111], so only the most recent data from molecular analyses will be discussed. The aim is to summarise results from DNA sequencing studies and to integrate these data with other genetic analyses. 2 Type 1 diabetes is a complex genetic disease Type 1 diabetes is a genetic disease because it runs in families. About 10% of Type 1 diabetic patients are familial and, on average, a first-degree relative of a diabetic has about a 6% risk of disease compared with a 0.4 % risk for a random member of the population [80, 111]. Genetically identical monozygotic twins are about 36 % concordant for disease [71], implying that environmental factors are important for development of Type 1 diabetes. Apparently gene effects are not enough to precipitate beta cell destruc-

34

J . A . Todd

tion. This is very different from CF, which is a single gene defect. Individuals who carry two defective alleles of the CF gene always have the disease phenotype. The genes that cause Type 1 diabetes are present in normals and, therefore, are referred to as susceptibility genes. They provide genetic susceptibility which, in the presence of "disease-associated" environmental factors leads to beta cell destruction. With the development of a detailed genetic map for the human genome, based mainly on DNA polymorphisms, the "reverse genetics" approach is to find linked markers to disease genes by scanning the genome for markers that cosegregate with disease in families. There are many published examples of successful disease gene linkage studies, mostly for single gene defects [61, 121] but also for complex diseases such as atopy [20]. Another approach is to select candidate genes based on a knowledge of disease pathogenesis [21]. Even though the region of DNA that contains the CF gene was identified by linkage analysis in families, biochemical information about the disease was essential. The defective gene was suspected to be expressed in sweat glands and function in ion transport. Candidate DNA segments in the region of chromosome 7 that were closely linked to disease were used to screen a sweat gland cDNA library. This step provided important evidence that a particular DNA segment actually was the CF gene [48]. Until very recently, the chosen strategy for genetic analysis of Type 1 diabetes has been the candidate gene approach. Two candidate genes, HLA class II genes in the MHC on chromosome 6 and the insulin gene on chromosome 11 have yielded positive and reproducible results, indicating that these genes influence susceptibility to Type 1 diabetes. Based on the evidence that the juvenile-onset, insulin-dependent kind of diabetes might be an autoimmune disease and the fact that immune responses are controlled by genes within the MHC, MHC genes were, therefore, logical and attractive candidate disease susceptibility genes. The disease and its complications are a result of beta cell destruction and insulin deficiency. The insulin gene itself may influence susceptibility of beta cells to immune destruction. It is possible that insulin is a key autoantigen in disease development. Susceptibility to Type 1 diabetes has been associated with certain alleles of both the insulin gene and several MHC genes. Reported associations of other genes with disease appear to be either very weak or not real [21 ]. The contributions of the insulin gene region and the MHC appear to be very different, ranging from 20 % - 6 0 % for the MHC and minor for the insulin gene [80, 85, 103]. 3 MHC class II genes are associated and linked to susceptibility genes for Type 1 diabetes

The first typing reagents for MHC antigens identified the HLA class I alleles HLAB15 and -B8 as associated with susceptibility to Type 1 diabetes in population studies [65, 66]; (Fig. 1). With the development of cellular and serological typing methods for alleles of the class II loci, the strongest population associations are now known to be with the class II region (Fig. 2; [107, 111]). Over 90% of caucasian Type 1 diabetics are DR3 or DR4 positive compared to 4 0 % - 5 0 % of controls. Many class I and class III alleles are in strong linkage disequilibrium

Genetic analysis of susceptibility to Type 1 diabetes class

DPB2

i

II

class

DRA 2 1 B C2

I

II

III

class

35 I

TNF B C

III

IK0p 510 10100 1 oo 20r00 251oo 30100 3 100 Fig. 1. Physical map of the human major histocompatibility complex. Only the most centromeric and telomeric loci are shown for each of the three regions e.g. DPA2 and DRA for the class II region. The number of expressed genes and pseudogenes varies according to the haplotype. The class II region has at least 7 expressed genes, the class II region has 7 expressed genes that include the TNF-c~ and -/3 genes, plus at least 13 more between C2 and TNF-a, including two HSP-70 loci. The class I region contains three major expressed loci plus at least 15 other related genes, whose expression is different from the HLA-A and -B loci. Data is from [12, 24, 89, 104, 117] and other references cited in the text

r

c,l

0 Kbp

1000

1

.I

Fig. 2. Detailed physical map of the MHC class II region. Expressed genes: black; pseudogenes: white; genes of undefined status: cross-hatched. Figure taken from [117]

with certain class II alleles. Associations with class III or class I genes are, therefore, probably secondary to class II associations. In addition, linkage analysis -in families also indicates that an important susceptibility gene is located within the class II region [7, 103]. This does not exlude the possibility that susceptibility genes exist telomeric to DRA; current data suggest that these influences are less important than class II region effects. Evidence that class II-linked susceptibility determinants do not act independently of other MHC genes comes from the observation that only subsets of DR-DQ haplotypes predispose to disease. Other interpretations of the data from the analysis of the associations of haplotypes with Type 1 diabetes are possible (see Section 14). Despite the fact that Type 1 diabetes is a common disease and many multiplex families have been studied, relatively little progress has been made towards identification of the susceptibility gene within the class II region using linkage analysis. This is due mainly to infrequent recombination between MHC loci, particularly between HLA-B and HLA-DQ. No recombination between DR and DQ has been reported in any HLA-typed family. Susceptibilty to beta cell destruction in human,

36

J.A. Todd

rat [19, 42] and mouse [35, 77, 122] is linked to the MHC region. Genetic mapping in the rat has shown that susceptibility maps centromeric to the class I region, towards the class II region. Because no class I/class II recombinants have been described in a NOD cross with a diabetes resistant strain, susceptibility in the mouse can only be mapped to the entire MHC. Linkage an segregation and HLA antigen frequency analyses have been performed on a large body of combined HLA-typing data from 1792 diabetic families [111] and also on data from HLA and insulin gene restriction fragment length polymorphism (RFLP) mapping in 94 families [103]. Conclusions from these studies include: (1) at least one gene that influences susceptibility to Type 1 diabetes is very closely linked to the DR-DQ subregions; (2) the DR genes are unlikely to be the susceptibility loci; (3) a simple single gene, two allele model cannot account for the data. Two genes with at least two alleles each appear to be a minimum requirement to explain HLA-linked susceptibility; (4) DR3 haplotype-associated susceptibility (in the absence of DR4) appears to be recessive due to an observed excess of DR3 homozygotes; (5) DR4 haplotype-associated susceptibility (in the absence of DR3) appears to be dominant due to an observed excess of DR4/X heterozygotes and also because the proportion of diabetic parents who transmit DR4 to diabetic offspring is significantly higher than the gene frequency of DR4 in the overall diabetic population [55]; (6) because DR3/4 heterozygotes are at greater risk of disease than the sum of the risks for DR4 and DR3 homozygotes, it is concluded that the susceptibility determinants on DR3 and DR4 haplotypes are separate but interactive. DR4- and DR3-associated susceptibility could be due to different alleles of the same gene or to alleles of different genes; (7) the high risk for first-degree relatives cannot be totally explained by HLA-linked genes. Other genes outside the MHC, presumably on other chromosomes probably influence susceptibility. One caveat to this conclusion is that a large part of familial aggreation could be due to sharing of environmental factors. The available data cannot distinguish between these two possibilities. Further evidence that human Type 1 diabetes is polygenic comes from the fact that the rat and mouse diseases are definitely the result of more than one susceptibility gene. Finally, (8) the insulin gene or a closely linked gene influences susceptibility to Type 1 diabetes.

4 The contribution of MHC-linked genes to susceptibility Based on serological HLA-typing data, estimations of the contribution of MHClinked genes to susceptibility vary. The concordance rate for MHC-identical siblings is about 13%. It may be as high as 20% if DR3 and DR4 haplotypes are shared [111]. The difference between 13 % risk for MHC-sharing siblings and 36% risk for monozygotic twins indicates that other genes are important. Since familiy members may share environmental factors more often than random members of the population, the difference between 13% and 36% may not be as prone to environmental effects as the difference between family risk (6%) and risk for a random individual (0,4 %). Calculations based on the number of affected siblings that share both MHC haplotypes, monozygotic twin concordance rate and the risk

Genetic analysisof susceptibilityto Type 1 diabetes

37

for MHC-identical siblings indicate the MHC contributes about 60 % of the genetic component of susceptibility to Type 1 diabetes [85]. Calculations of the contribution of MHC-linked genes to susceptibility based on a comparison of the risk for siblings that share neither MHC haplotype (risk=l.8%), and the risk for an individual member of the population (0.4%) indicate that the contribution of the MHC is small (less than 20%) [80]. It is reasoned that the high level of MHC haplotype non-sharing in diabetic families (7%), must be the result of at least one other major disease susceptibility locus. Aside from possible environmental effects, this calculation does not take into account the possibility that siblings that do not share MHC haplotypes as detected by HLA serology, actually do share different MHC-linked susceptibility determinants that are not apparent from serological typing information. It is possible that a high proportion of the risk of disease of MHC-non-sharing individuals is due to MHC susceptibility gene effects. This point is discussed in more detail later (Sections 9, 11 and 14) in the context of how different alleles of the DQA1 and DQB1 loci might account for the association of MHC with disease susceptibility.

5 Type 1 diabetes is an autoimmune disease The evidence that Type 1 diabetes involves an autoimmune destruction of pancreatic beta cells has been reviewed [15, 25, 84]. An early event, and probably an essential one, in disease development is the infiltration of islet cells by T and B lymphocytes and macrophages, called insulitis. Recent experimental evidence from an animal model of Type 1 diabetes, the non-obese diabetic (NOD) mouse demonstrate that cells of the infiltrate participate in beta cell destruction. Beta cell destruction is T cell dependent because the disease can be transferred from diabetic animals to healthy recipients by the injection of T cells [57]. Diabetic T cells are, therefore, sufficient to cause beta cell destruction, although the recipients either have to be neonatal [10] or irradiated [57]. Both CD4 and CD8 subsets are required [57]. CD8 T cells, isolated from NOD islets, can be grown in culture as T cell lines and are able to lyse beta cells. Lysis is MHC restricted and blocked with anti-Kd antibodies [60]. Beta cell destruction may, therefore, be T cell mediated. T cell clones have been isolated from NOD islets that are islet cell specific and I-AN~ [34, 78]. Both CD4 and CD8 T cell clones are required for insulitis [78]. It is not clear how efficient these T cell clones are in transfering disease to healthy recipients [78]. Clearly, cloning of T cells that are pathogenic in vivo is an important step towards the identification of beta cell autoantigens. A simple scheme for immune pathogenesis, therefore, involves CD4 T cell activity providing (cytokine) help to CD8 T cells which lyse beta cells. This model does not exclude roles for other immune system-related effects, such as lysis of beta cells by macrophages [3, 120], by exposure to high levels of cytokines such as IL-1 [67], by lysis by CD4 T cells or by a combination of the latter. An essential role for CD4 T cells in immunopathology provides a strong rational for considering

38

J.A. Todd

class II genes of MHC as primary candidates for MHC-linked disease susceptibility genes.

6 MHC class II genes as candidate disease susceptibility loci Class II gene products determine T cell activation in response to antigen [93]. T cell antigen receptors can only recognise antigen, in the form of peptide fragments, when it is bound to a class II molecule [91, 93]. Class II (and class I) genes are highly polymorphic. Most class II polymorphism encodes structural variation in the peptide-binding cleft of the molecule [16]. The peptide-binding site is formed by folding of the a and ~ chains into a single domain which, with bound peptide is, the ligand of the T cell receptor. Polymorphic residues can be considered as active site residues in peptide binding. A large proportion (70%) of the ability of individuals to respond to different peptides is determined by the capacity of the individual's class II molecules to bind peptides. In general nonbinders are non-responders [91]. Both the magnitude and specificity of immune responses are, therefore, dependent on MHC class II genotype. The specificity of T cell antigen receptors that emerge from the thymus also depends upon class II (and class I) genotype. Certain T cell receptor o~,~ combinations are either selected [109] or eliminated [47] after recognition of MHC molecules complexed with self antigens in the thymus. T cell selection can account for instances where class II peptide binding occurs but the animal is a nonresponder. More recently, T cell tolerance induced in the periphery has also been associated with MHC class II (and class I) recognition [93]. Immune responsiveness to exogenous and self antigens is, therefore, closely associated with class II polymorphism. Class II molecules that bind the autoantigen might well be expected to be positively associated with disease susceptibility. Data from DNA sequencing studies indicate that DQ molecules predispose to disease in such a fashion.

7 HLA typing at the DNA level In 1983, Owerbach and colleagues [72] made the important observation that DR4-positive Type 1 diabetics and DR4-positive controls differed in the frequency of certain DRB-l-associated RFLP. These polymorphisms are due to different alleles at the DQB1 locus [49]. Other RFLP studies confirmed this result and showed that, for at least three different DR haplotypes (DR2, DR4 and DR6) variation at the DQB1 locus was more strongly associated with disease than variation at the DRB1, DRB3, DRB4 or DRA loci [4, 13, 18]. Based on these allelic associations it was concluded that a gene influencing susceptibility to Type 1 diabetes lies in the DQ subregion, a stretch of DNA of about 150-250 kbp. Because beta cell destruction is CD4 T cell dependent, it was reasoned that the expressed DQ loci, DQA1 and DQB1 (DQA2 and DQB2, formerly DXo~ and DXr are probably not expressed) were primary candidates for disease genes. Identification of the CF gene involved DNA sequencing of segments of DNA that were most closely

Genetic analysis of susceptibility to Type 1 diabetes

39

linked (family studies) and associated (population studies) with disease. Exactly the same approach was used to test the hypothesis that the DQ genes were susceptibility genes for Type 1 diabetes. DQ and DR alleles from patients and controls were cloned and sequenced [39, 113], using the polymerase chain reaction (PCR; [87]). Progress in identifying the loci responsible for MHC-linked susceptibility to disease has paralleled the ability to resolve polymorphisms. RFLP analysis defined which haplotypes were most important to analyse by DNA sequencing. This approach is still valid today, because RFLP detect polymorphisms well beyond the scope of most DNA sequencing projects and provide useful signposts for further analysis (see DR4 Dwl4 example in Section 14).

8 DQB1 polymorphism correlates with disease susceptibility DNA sequencing of DR and DQ alleles from patients and controls showed that polymorphism at codon 57 of the DQB1 locus correlated with susceptibility to Type 1 diabetes [39, 113]. DQB1 alleles that were associated with susceptibility by RFLP analysis [i.e. DQw6(AZH), DQw6(1.19) and DQwS] all encoded a noncharged amino acid at position 57 (Ala, Ser or Val) (Table 1). In contrast DQB1 alleles that were not associated with positive susceptibility encodes Asp57. The DNA sequencing approach also showed that DQ alleles in patients were no different in structure from normal alleles. This result contrasts with CF alleles, many of which have a missing codon (at position 508) and are mutant alleles [48]. DQ alleles, if they are primary determinants of disease, are susceptibility alleles. They are common in both patients and controls. The importance of position 57 in DQ structure and function is evident from several observations. Both antigen-specific [105] and alloreactive [52] T cell recognition is sensitive to polymorphism at this site. DQ /3 chains have been described that differ only at amino acid 57 in the polymorphic N-terminal domain, e.g. DQw8 and DQw9, DQw5 (1.1) and DQw5 (AZH), and DQwS(1.1) and DQwS(1.9) [39,113]. The DQw5(1.9) allele is very strongly associated with susceptibility to the autoimmune skin disease phemphigus vulgaris [92, 101]. This supports the hypothesis that a single residue can influence susceptibility to autoimmune disease, presumably through a change in peptide-binding specificity and/or T cell recognition. In a hypothetical model of class II structure based on the crystal structure of a class I molecule,/3-chain residue 57, if it is Asp (which has a negatively charged side chain), forms a salt bridge with Arg79 of the ot chain [16]. If this is confirmed in a class II crystal structure, then residue 57 could affect the structure of the peptide-binding site. The NOD mouse has a unique I-A/3-chain structure with Set57. All diabetesresistant strains have Asp57 in the I-A /3-chain [1]. Hence it appears that the putative diabetes-associated defect in the DQ/3 chain is conserved between mouse and man. Sequences of the DR and DQ homologues from the diabetic BB rat and diabetes-resistant strains also support the correlation. All rat class II /3-chain sequences have Ser57 [17]. It is noted that none of these alleles, including those from diabetes-resistant strains, encode Asp57. The absence of/3-chain Asp57 in

40

J.A. Todd

Table 1. HLA-DR and DQ haplotypes and their associations with Type 1 diabetes in blacks and caucasians DRB 1

DQB 1

DQA 1

DQ~-chain residue 57

Disease association

DR2 DR2 DR2 DR4 DR4 DR6 DR6 DR6 DR1 DR3 DR3 DR5 DR7 DR7 DR8 DR9 DR9

DQw6(1.2) DQw6(1.12) DQw6(1 .AZH) DQw7(3.1) DQw8(3.2) DQw6(1.18) DQw5(1.9) DQw6(1.19) DQw5(1.1) DQw2 DQw4 DQw7 DQw2 DQw2 DQw4 DQw9(3.3) DQw2

A1 A1 A1 A3 A3 A1 A1 A1 A1 A4 A4 A4 A2 A3 A4 A3 A3

Asp Asp Ser Asp Ala Asp Asp Val Val Ala Asp Asp Ala Ala Asp Asp Ala

N + N + N + + + N N N + N N +

Each haplotype has a different association with susceptibility to Type 1 diabetes [27, 39, 82, 83, 111, 113]. The assigmnents shown are, therefore, only approximate N, Neutral with respect to Type 1 diabetes susceptibility, implying that the haplotype is generally neither significantly positively or negatively associated with susceptibility. Also there is variation between studies. +, positive, where an increased number of patients are positive for these haplotypes than controls in more than one study and in more than one population. - , negative, where more than one study finds a decreased number of patients with the haplotype compared with controls. There are at least three different A1 and A4 alleles with a small number of amino acid differences in the N-terminal domains between members of the A1 and A4 families [32]. There are also multiple subtype alleles of DR1 ,DR2,DR3,DR4,DR5 and DR6 alleles of the DRB1 locus. Except for DR4 subtypes, the associations of others have not been studied in detail with respect to susceptibility to Type 1 diabetes and are not discussed

diabetes-resistant rat M H C haplotypes is consistent with the fact that MHCBB/MHCDiabet . . . . . . i s t a n t a n i m a l s develop diabetes at a level and severity that is only slightly less than M H C BB homozygotes [19]. This is an important difference b e t w e e n the genetics of the rat disease c o m p a r e d with the m o u s e and h u m a n diseases, in which M H C heterozygotes (resistant M H C / p e r m i s s i v e M H C ) have a five-to ten fold reduced p r e v a l e n c e of disease [39, 58, 83, 113, 124].

9 Disease gene mapping using recombination events between DR and DQ L i n k a g e analysis within the class II region is o f little use because of infrequent r e c o m b i n a t i o n b e t w e e n loci in this region. A similar p r o b l e m was e n c o u n t e r e d in the region of D N A a r o u n d the C F gene. T h e single codon deletion in the C F gene is found mostly on one haplotype. H e n c e resolution o f the exact location o f the C F gene by linkage analysis was limited to an area where there was signifi-


41

cant linkage disequilibrium between neighbouring alleles. Because a DR-DQ crossover event in a family has not yet been documented, linkage disequilibrium also prevents fine mapping in the class II region. Most DRB1 alleles are in strong linkage disequilibrium with only one DQB1 (and DQA1) allele. It is difficult, therefore, to distinguish between DR effects and DQ effects on disease susceptibility. In different populations, however, DR alleles are in linkage disequilibrium with different DQ alleles. This is also true of certain caucasian haplotypes and comparative analysis of these haplotypes has been used to understand the evolution of haplotypes [31] and also used to map susceptibility to Type 1 diabetes [63]. The existence of race-specific recombinant haplotypes offers the opportunity to analyse the association of a single DQB1 (or DQA1) allele in the presence of more than on DRB1 allele. RFLP analysis of black DR-DQ haplotypes by Barnett and colleagues [27] has identified four race-specific DR-DQ haplotypes (Table 1) that map susceptibility centromeric to DRB1 and support the hypothesis that not only does DQB1 determine susceptibility to Type 1 diabetes but also that DQA1 is important. The associations of most MHC haplotypes are consistent with DQB1 codon 57 correlation [113]. There are, however, DQ allele associations with Type 1 diabetes that cannot be explained by codon 57 [39, 113]. Since the structure [16] and funtion [88] of the ot and/3 chains are integrally associated, it was predicted that the DQ o~ chain would also correlate with susceptibility. The DQB1 allele DQw2 (Ala57) is positively associated with susceptibility on DR3 haplotypes but is only neutral on DR7 haplotypes [111]; Table 1). This implies that the DQB1 locus is not the only susceptibility determinant, and raises the possibility that DQB1 is only a closely linked marker for the disease susceptibility gene in this region. Fletcher et al. [27] described a race-specific DR7 haplotype in a black population that, in contrast to caucasian DR7 haplotypes, was highly diabetogenic. The Nterminal domain-encoding sequences of the DRB1, DRB4 and DQB1 loci on this predisposing DR7 haplotype found in blacks were identical to those found on caucasian DR7 haplotypes. The only coding sequence differences were at the DQA1 locus: the usual allele, A2 was replaced with the A3 allele (Fig 3; [115]). The A3 allele is also found on both black and caucasian DR4 haplotypes (Table

DQBI

DQA1

DRB1

DRB4

DRA IDDM

DQw2 A 2 c e n t r o m e r e ~

DQw2 c e n t r o m e r e ~-i-..i',.i'.('..iJ

A 3

DR7 ~ ~ i . i i . i "

association

telomere

caucasian

neutral

e l o rn e r e

black

positive

DR7 I . i . . i - ~ . i . . : ~ t

Fig. 3. Comparative analysis of DR7 haplotypes in blacks and caucasians with respect to Type 1 diabetes susceptibility. Details given in Section 9 [27, 115]. Replacement of the A2 allele of the DQA1 locus for the A3 allele on the DR7 haplotypes correlates with a change in the association of DR7 with Type 1 diabetes from neutral to positive. Dotted boxes are likely to have identical DNA sequences or only minor differences

42

J . A . Todd

1) and since DR4 haplotypes carry the most potent susceptibility alleles for Type 1 diabetes, the A3 allele is a strong candidate for a positive susceptibility determinant. This result also implies that the DR7-associated allele, A2 of the DQA1 locus confers resistance to Type l diabetes [73]. A3 in combination with the DQw2 allele of the DQB1 locus, therefore, correlates with susceptibility to Type 1 diabetes on DR7 haplotypes. A3 and DQw2 also occur on race-specific, black DR5 [56] and DR9 [28] haplotypes, both of which predispose to disease. Allelic sequencing of a diabetogenic black DR9 haplotype showed that the neutral allele DQw9 (Asp57) is replaced with DQw2 (Fig 4; [115]). The A3/DQw2 allelic combination (on the same chromosome) is very rare in caucasians. Owerbach et al. [73] have described an A3/DQw2 haplotype in a caucasian Type 1 diabetic. The A3/DQw2 molecule is common in caucasian Type 1 diabetics, however, because 30 % - 4 0 % of patients are DR3/4 heterozygotes (versus 3% of controls; [111]) and the DR3 and DR4 haplotypes have the DQw2 and A3 alleles, respectively. Transcomplementation between DQ alleles on different chromosomes is one explanation for the observed increased risk of DR3/4 heterozygotes [62, 102, 107]. Although allelic sequences have not been published from DR3/9 Chinese heterozygotes, who are also at increased risk of Type 1 diabetes [34], they may also have the A3/DQw2 pair, encoded in trans. Finally, it is noted that the DR3/4 effect is not as strong in blacks as it is in caucasians [27, 126]. An explanation for the lack o f an apparent DR3/4 effect in this population is that over 50 % of patients are positive for a A3/DQw2 haplotype [56]. The DR3 allele in black populations is associated with two DQB1 alleles, DQw2 and DQw4 [27, 40]. The frequencies of these haplotypes are about equal in controls, but almost all Type 1 diabetics have the DR3,DQw2 haplotype (Fig 5; [27]). These results support the DQB1 residue 57 correlation because DQw4 has Asp57, whereas DQw2 has Ala57. A caveat to this comparison is that these two haplotypes have many other differenes, including minor differences (two to four amino acids) in both the DQA1 and DRB1 loci [41]. Any one of these other differences could account for the different associations of these two DR3 haplotypes. In general, therefore specific DQA1 and DQB1 alleles correlate with diesease susceptibility to Type 1 diabetes despite variation at the DRB1 locus. The suscepDQB 1

DQA 1

DQw9 el

A 3 I :.:'i.i'i.ii.i ii

DRB 1

DRB4

DRA I D D M association

centromere

DQw2 A 3 c e n t r o m e r e ~

DR9 L ' . I . I . I . I . ? I . I ~

t e Io m e r e

caucasian

neutral

telomere

black

positive

DR9 ~ . . . -

-.:.~-. : .

Fig. 4. Comparative analysis of DR9 haplotypes in blacks and caucasians with respect to Type 1 diabetes susceptibility. Details given in Section 9 [27, 115]. Substitutionof the Asp57-positive DQ /s-chain allele, DQw9 for the Asp57-negative DQ/S-chain allele, DQw2 correlates with a change in the association of DR3 with Type 1 diabetes from neutral to positive. Dotted boxes are likely to have identical DNA sequences or only minor differences

Genetic analysis of susceptibility to Type l diabetes DQB1

DQA1

DRB1

DRB3

43

DRA IDDM association

DQw2 A4 c e n t r o m e r e ~

centromere

DQw4

A4 [..i...i...?..?..I

DR3 ~ ~ ' . : ' i " :

DR3 [.,i...i...:~. :

.

.

.

~

telomere

t

el o m e r e

caucasian & black

positive

black

neutral

Fig. 5. Comparative analysis of DR3 haplotypes in blacks and caucasians with respect to Type 1 diabetes susceptibility.Detailsgiven in Section9 [27]. Replacementof an Asp57-positiveDQ/S-chain allele, DQw4 for an Asp57-negativeDQ ~3-chainallele, DQw2 on DR3 haplotypescorrelates with a change from neutral to positive with respect to susceptibilityto Type 1 diabetes. It is noted that the shadingdoes not mean that the alleles are identical. There are coding and non-codingdifferences between these two haplotypes, includingminor coding sequence differencesat the DQA1 and DQB1 loci [41] tibility locus (or loci) that lies in the DQ subregion appears to act largely independently of DRB1. This conclusion is consistent with other genetic analyses that indicate that DRB1 itself in unlikely to be the susceptibility gene for Type 1 diabetes [103, 111].

10 DQ allele associations in the Japanese Further support for a role for the A3 allele in Type 1 diabetes susceptibility comes from analysis of DQ and DR alleles present in Japanese controls and patients [116]. The strongest population association with disease was with the A3 allele (relative risk= 19.7; P < 0.001). The DQw4 allele had a weaker association with disease than the A3 allele (relative risk =4; P =0.008), implying that susceptibility is determined by a gene that lies closer to DQA1 than DRB1. Because the A3 allele is associated with positive susceptibility in three different populations (caucasian, black and Japanese) and on several different haplotypes (that is in the presence of different DRB1 alleles and also different alleles at other MHC loci), it is reasonable to propose that the A3 allele itself predisposes directly to disease [ 116]. Furthermore, the activity of A3 in disease development appears to be dependent on DQB1 polymorphism because A3 paired with Asp57 DQB1 alleles (e.g. DQw7) is neutral for disease in caucasians ([64, 81]; Nepom, personal communication; and Stastny, personal communication). The Japanese analysis raises a further point. In Japan the DQw9 and DQw4 alleles, which encode Asp57, are positively associated with Type 1 diabetes [2, 11, 116]. It is argued [116] that because Japan has the lowest incidence of Type 1 diabetes in the world [23], the DQw9 and DQw4 alleles are only weakly predisposing to disease. In caucasian populations, which have high frequencies of highly predisposing haplotypes [107], Asp57-postive molecules, DQw7 and DQw9 are neutral ([54, 111]; Nepom, personal communication; and Stastny, personal communication). It appears that the putative role of A3 in Type 1 diabetes correlates with DQB1 polymorphism, particularly codon 57. If this interpretation

44

J.A. Todd

is correct, then Asp57 of the DQ/3 chain correlates with reduced susceptibility to Type 1 diabetes in all three populations studied. Recently, the Chinese have been shown to have a high frequency of Asp57 DQB1 alleles and they also have a low incidence of Type 1 diabetes [6]. The frequencies of DQA1 and DQB1 alleles in a population may determine the incidence of Type 1 diabetes [6, 116]. Confirmation of this hypothesis will require analysis of the frequencies of DQ alleles in different ethnic groups living in the same area, with accurate ascertainment of Type 1 diabetes incidence. One possible interpretation of the Japanese and Chinese data is that important environmental disease-triggering factors are absent in these countries. Ascertainment of disease incidence of Japanese and Chinese living in other countries where the incidence of disease is high (e.g. USA) would be informative.

11 A mechanism for the association of the MHC with Type 1 diabetes

Sequencing of alleles of class II genes and population-specific association of DQ alleles with disease susceptibility indicate that the DQA1 and DQB1 loci contribute directly to susceptibility to Type 1 diabetes. Combined analysis of family data suggest that there are at least two MHC-linked susceptibility loci and that the DR4-associated determinant is the most diabetogenic [26, 55] and is inherited in a dominant/intermediate fashion [55, 111]. It is proposed that the A3 allele of the DQA1 locus is a strong candidate for the DR4-associated positive susceptibility determinant and that Asp57-negative alleles of the DQB1 locus are permissive for disease development [81]. Asp57 DQB1 alleles correlate with reduced susceptibility (not dominant protection). DR3-associated susceptibility appears to be recessive and it is suggested that the DQw2 allele is one of the determinants involved. It is noted that only 5 % - 1 0 % of caucasian patients are DR3/3 homozygotes [111], and that they may have a less severe form of disease [51]. It has been suggested that DR3-associated susceptibility, in the absence of DR4, is weak compared with the DR4-associated susceptibility [55] and may have an enhancing effect on DR4 haplotype-associated susceptibility. Since the Asp57-negative I-A molecule (the murine homologue of DQ) is the only expressed class II molecule in the NOD mouse (the NOD mouse does not express I-E), it is likely that it is responsible for autoantigen presentation to CD4 T cells. Disease incidence in NOD backcross mice that are heterozygous at the MHC (Asp57 I-A/non-Asp57 I-A) is greatly reduced [124]. This may be due to a reduced capacity for autoantigen presentation because there are less non-Asp57 I-A molecules available. The Asp57 I-A molecule may also actively reduce the autoimmune T cell response through mechanisms such as clonal deletion, competition for peptides or T cell suppression. The relative roles of different I-A (and DQ) alleles in autoimmunity is a major area of future research (see Section 13). By analogy with the mouse, DQ molecules, particularly the A3/DQw2 o~,/3 heterodimer, may be responsible for presentation of autoantigen to CD4 T cells in humans. The DQw2 allele on DR3 haplotypes may enhance the putative effects of the A3 allele in disease by increasing the magnitude of an autoimmune response. Individuals who are homozygous for A3 and Asp57-negative alleles, particularly,


45

DR4 homozygotes, are at greater risk of disease compared with DR4/X subjects (X is not DR3 or DR4) because they have a higher density of antigen-presenting DQ molecules and, therfore, can produce a stronger autoimmune response. Heterozygous individuals (Asp57/non-Asp57 DQ ~ chain) are at greatly reduced risk (five-to tenfold) of Type 1 diabetes [58, 83, 113, 116]. The presence of Asp57 DQB1 alleles may reduce the efficiency of autoantigen presentation by causing a reduction of the number of expressed molecules capable of presenting the autoantigen efficiently. It is emphasised that there are no unique class II sequences in patients and, hence, diabetogenic alleles are not sufficient to cause Type 1 diabetes. MHC-linked susceptibility alleles must act in concert with other genes that normally prevent autoantigen presentation leading to beta cell destruction. In the NOD mouse and BB rat there is experimental evidence for: (a) defects in bone-marrow derived cells, which may function as antigen-presenting cells in the thymus [9, 123]; (b) defects in macrophage function [96], which may or may not be related to class II structure; and (c) insulitis in the NOD mouse, which appears to be only partly controlled by MHC genes, and at least one other gene, behaving in a dominant fashion and not linked to the MHC, which determines the development of insulitis [ 122].

12 DR2 haplotype-encoded resistance One of the most striking HLA associations with Type 1 diabetes is the negative association of the DR2 allele [ 111]. DR2 haplotypes, therefore, provide resistance to disease, particularly when susceptibility is encoded by DR4 haplotypes. Not all DR2 haplotypes are negatively associated with Type 1 diabetes. There is at least one diabetogenic DR2 haplotype (called DR2 DQw6(AZH)) [4, 13,18,26]. Consistent with the codon 57 DQB1 correlation, this DR2 haplotype is the only one to encode a non-Asp57 DQ ~ chain (Table 1). Other DR2 haplotypes encode Asp57 DQ ~ chains. RFLP analysis also maps susceptibility and reistance to the DQ subregion in different ethnic groups [28, 50, 68, 95]. A DQB1 RFLP that marks resistance to Type 1 diabetes on DR2 haplotypes also correlates with resistance on DR6 haplotypes [27, 28, 50, 94, 95]. As described above for putative susceptibility alleles of DQA1 and DQB1, DQassociated sequences correlate with resistance across ethnic group variations in haplotypes and also in the presence of different DRB1 alleles. The structures of the two DQ molecules encoded by the protective DR2 and DR6 haplotypes are very similar (differences are at/3-chain residue 30, and o~-chain residues 25 and 41; [39, 113]. It is reasonable to assume that susceptibility and resistance might be encoded by alleles of the same loci and that interactions of the products of these alleles determine the degree of susceptibility. However, despite this strong correlative evidence that the DQw6 molecule encoded resistance to Type 1 diabetes, it is possible that resistance is encoded by a gene that is closely linked to the DQ loci. DR5 haplotypes are also significantly less frequent in patients [54, 111]. Associations of DQ alleles can also explain this observation. Caucasian DR5 haplotypes almost always have DQw7 (Asp57), which is neutral with respect to

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disease on DR4 haplotypes. DR4 haplotypes also have the A3 allele, which may predispose to disease. A3 in the presence of the Asp57 DQw7 allele is only neutral (see Section 10). DR5 haplotypes, in contrast to DR4 haplotypes, do not have the A3 allele but carry the A4 allele of the DQA1 locus. Therefore, DR5 haplotypes lack both the putative susceptibility determinants, an non-Asp57 DQB1 allele and the A3 allele. In blacks, replacement of the DQw7/A4 alleles with the DQw2/A3 alleles on DR5 haplotypes correlates with a change of association with disease from negative to positive [56]. This supports the hypothesis that variation at DQ determines the association of the DR5 haplotype with disease.

13 Possible mechanisms for DR2-associated resistance

The rational for considering DQ and DR molecules as strong candidates for susceptibility genes is that they are highly likely to be involved in autoantigen presentation. Their ability or inability to bind autoantigen could determine susceptibility or resistance. Interestingly, the DR2-associated effect appears to be dominant [ 113]. Lack of autoantigen presentation by the DQw6 molecule may not be a sufficient explanation for the observed decrease in DR2 haplotypes in patients. The DR2-associated DQw6 antigen, in particular the Dwl2 subtype, has been shown to mediate immune suppression effects on DR-restricted responses to a schistosomal antigen [90]. Nonresponsiveness to Mycobacteriumlepraeand Streptococcal cell wall antigen and other immune response effects also appear to be controlled by the DQ subregion [70, 90]. Recently, low responsiveness to the early expressed antigens of human cytomegalovirus (CMV) has been associated with the DQw6 antigen, both on DR6 and DR2 haplotypes (Lagaaij et al, unpublished). This observation is interesting because CMV infection has been associated with Type 1 diabetes [75] and also, CMV infects pancreatic beta cells [44]. Low responsiveness to CMV may be related to resistance to Type 1 diabetes in that DQw6positive individuals are unable to develop strong immune responses against CMVinfected beta cells and, therefore, fail to precipitate immune damage that, in DQw6-negative individuals, may help trigger beta cell destruction. The antibody response to schistosomal antigen involves DQw6-restricted T cell suppression, which depends on both CD4 and CD8 T cells. The data indicate that DQw6-restricted CD4 T cells help CD8 T cells suppress the antibody response [38]. Results from experiments in the mouse indicate that CD4 T cells can suppress antibody responses by lysing antibody-producing B cells [99]. Mechanisms have been established therefore that might explain the association of DR2 haplotypes with Type 1 diabetes. Evidence from studies in the NOD mouse suggest that T cell suppression is important in the development of Type 1 diabetes [ 14]. The suppression that results from in vivo anti-class II treatment of NOD mice depends on the activity of CD4 T cells [14]. Transfer of these cells into NOD mice prevents the transfer of disease on subsequent injection of spleen cells from diabetic mice. NOD mice less than 4 weeks old are also refractory to disease induction by diabetic spleen cell transfer [10, 57]. It appears that certain T cells can actively inhibit the autoimmune response. Recently, T cell lines have been isolated from NOD mice that proliferate

Genetic analysis of susceptibilityto Type 1 diabetes

47

in the presence of NOD spleen cells, termed autoreactive T cells [79]. The response of T cells to syngeneic spleen cells is called the syngeneic mixed lymphocyte reaction (SMLR). Injection of these autoreactive T cell lines into NOD mice prevents the development of disease [79]. Perhaps the presence of such T cells in normal NOD mice protects against the development of disease. Serreze and Leiter [96] have found a defect in the NOD SMLR and speculated that this might be partly responsible for the development of disease. A second possible mechanism that might account for the negative association of DR2 haplotypes with Type 1 diabetes is clonal deletion of beta cell-reactive T cells [76, 114]. During T cell development in the mouse, autoreactive T cells are eliminated in the thymus by negative selection [46, 76, 93]. Recognition of I-E molecules by developing T cells results in T cell death. Inhibition of clonal deletion in the thymus by cyclosporin treatment results in release of autoreactive T cells into the periphery and the development of organ-specific autoimmunity [43]. A defect in clonal deletion is, therefore, an attractive explanation for autoimmunity. Class II polymorphism could account for such a defect. Clonal deletion has not yet been demonstrated in human, although this might be due to a lack of antibodies that recognise specific T cell receptor V region families [47]. NOD mice are I-E negative. Crosses of NOD with the I-E negative, diabetesresistant strain B10 show that susceptibility to Type 1 diabetes maps to the MHC, irrespective of I-E genotype [ 122]. Insulitis and diabetes can be prevented in NOD mice, however, by expression of I-E in NOD by the introduction of a funtional I-Ea transgene [118]. One explanation is that I-E expression deletes T cells bearing certain V~ segments. It is possible that a class II molecule encoded by diabetesprotective DR2 haplotypes mediates resistance to Type 1 diabetes by a similar mechanism [114]. It is argued above from data of allelic associations with disease that this molecule is more likely to be DQw6 than DR2.

14 The role of non-class II MHC genes in susceptibility Another problem with the analysis of the MHC is that in addition to the strong linkage disequilibrium between alleles, many of the identified genes encode products that are involved with the immune response. Hence there are many excellent candidate genes! For example, class I gene products, which are the restriction elements of CD8 T cells, are probably involved in disease pathogenesis [60]. Expression of class I has been demonstrated in rat, mouse and human beta cells [29, 33]. It has been suggested that class I expression per se may be a causative defect in beta cell function that leads to diabetes [33]. Certain alleles of the complement genes encoded by the class III region are also strongly associated with susceptibility to diabetes [110]. The TNF genes are also encoded within the class III region (Fig 1). Administration of TNF-ot in vivo can prevent the development of diabetes in NOD mice. The association of alleles of complement and TNF genes must be analysed in different ethnic groups. It is possible that, despite the fact that linkage and population association data indicate a primary role for the class II region in suscep-

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tibility, class HI and class I alleles influence susceptibility. It is unlikely that class II genes funtion in immune responses in isolation. The phenotypic effects of MHC genes are probably the result of complex interactions. Support for a role for genes outside the class II region in Type 1 diabetes comes from analysis of HLA-DR and HLA-B genotypes in diabetic families. Only subsets of DR4 and DR3 haplotypes, defined by HLA-B typing, appear to be predisposing [112]. Some of these, notably the associations of the DQw7 and DQw8 subtypes of DR4 haplotypes with disease can be explained by variation at the DQ loci. However, others, such as the different associations with disease of HLA-B8, -B18 DR3 and non-B8,-B18 DR3 haplotypes suggest that non-class II genes influence genetic susceptibility. This is unlikely to be due to the B8 or B 18 alleles themselves because non-HLA-B8 o r - B 1 8 haplotypes can be diabetogenic [26]. Two groups have demonstrated recently that the DNA around the HLA-B locus and in the class III region contains a number of genes in addition to the established loci [89, 104]. The coding potential of this part of the MHC may be in the order of 3-4 genes per 100 kbp of DNA. These results bring new emphasis to the possibility that a susceptibility gene is closely linked to the known loci, but has not yet been identified. In the class II region there is sufficient DNA between the DQA2 and DQB1 loci and between the DQA1 and DRB1 loci to contain more genes (Fig 2). Additional loci are unlikely to be class II genes because the region has been thoroughly screened for class II-related sequences [12, 24, 102]. Identification of all coding sequences within the MHC remains an important prerequisite for an understanding of MHC disease associations. Also, for each coding sequence identified, polymorphic probes should be isolated. Two allele polymorphisms, such as the Nco 1 RFLP of the TNF-c~ gene [5] are of little value in determining the significance of an association of a certain region of the MHC with disease. Unfortunately, and in contrast to the mutant CF gene that is not found on normal chromosomes, any MHC gene that turns out to be a primary susceptibility determinant will also be present in normals; its contribution to genetic susceptibility will have to be verified by linkage studies in large numbers of families and analysis of allelic associations in several different populations. In this context it is noted that DR4,DR7 and DR9 haplotypes all contain an extra 110 kbp of DNA between DQA1 and DRB1 compared with DR3,DR5 and DR6 haplotypes [24]. The coding sequences of this segment remain to be investigated. It is unlikely, however, that this extra 110 kbp of DNA contains a primary determinant of susceptibility to Type 1 diabetes because DR7 and DR9 haplotypes also contain this segment of DNA and these haplotypes are not associated with susceptibility in caucasians. The associations of certain haplotypes in the absence of apparent variation at the DQ loci can be explained in another way. It is possible that there is variation at the DQ loci that is not within the structural parts of the genes, but lies in introns or flanking regions and that influences susceptibility to disease. Such polymorphisms might affect expression of alleles. If DQ alleles have direct effects on susceptibility then allele-specific expression would be predicted to be important. To date, no regulatory polymorphism has been described that is associated with susceptibility to any autoimmune disease. Hence, even though caucasian B8,DR3


49

and B18,DR3 and non-B8,B18 DR3 haplotypes all apparently contain the same DQ alleles, A4 and DQw2, minor sequence variations in promotor sequences, might, for example, be important for susceptibility. A potential candidate for such a polymorphism has been described for a DR4 haplotype. Two studies have shown that DR4,Dwl4,DQw8,A3 haplotypes are not as predisposing as DR4,Dw4,DQw8,A3 and DR4,Dwl0,DQw8,A3 haplotypes [4, 98]. Two other studies, however, have not found any significant difference between Dwl4 haplotypes and the other Dw DR4 haplotypes [74, 107]. Dw subtypes of DR4 haplotypes apparently differ only at the DRB1 locus [31, 39, 113]. The DQ alleles (DQw8 and A3) have the same sequences on Dwl4, Dwl0 and Dw4 haplotypes. Therefore, if the Dwl4 effect is real, then DQ polymorphism, as it has been currently defined, cannot explain the association of this haplotype with Type 1 diabetes. Perhaps, in this case, DRB1 is exerting an influence. An alternative explanation is that the DQ subregions of the Dw4, Dwl4 and Dwl0 haplotypes are different and it is these differences that account for the associations. Experimental support for this explanation comes from the recent identification ofa Dwl4-associated DQA1 RFLP that is not found an Dw4 or Dwl0 haplotypes [37]. The sequence polymorphisms responsible for, and associated with this, RFLP are not known but it is conceivable that they influence DQA1 expression. If DQA1 and DQB1 really are primary susceptibility genes then polymorphisms that affect allele expression and are associated with disease susceptibility should eventually be identified. This area of research is just beginning and may be a more rewarding line of investigation now that strong candidate genes for susceptibility loci have been identified. Two other haplotypes, DR8 and DR1, have been discussed and both haplotypes are weakly associated with disease in caucasians [54, 82, 111, 125]. The DR1 association is consistently found in caucasians. Significant DR1 effects in Type 1 diabetes have not been confirmed in studies of other ethnic groups [27, 28, 50, 68, 95]. An explanation of this may be that the number of patients studied has not been large enough to detect a weak, but positive association. This weak association is compatible with the DQ/3-chain residue 57 which is Val on DR1 haplotypes (Table 1). It has been reported that DR1/4 heterozygous Type 1 diabetics have a normal distribution of DQw7- and DQw8-positive DR4 haplotypes [108]. The frequency of DQw8 is usually increased in patients compared with controls. This result has not, however, been confirmed in one other report [74]. DR8 haplotypes are only increased significantly in frequency in the presence of DR4 haplotypes in Type 1 diabetics compared with controls, implying an interaction between DR4- and DR8-associated alleles. In this case, DQw8 is still increased in frequency in patients compared with controls [82, 83]. Caucasian DR8 haplotypes mostly have the DQw4 allele (Asp57), in association with the A4 allele of the DQA1 locus [32, 82, 83]. Perhaps the combination of A3 (from the DR4 haplotype) and DQw4, which is probably common in Japanese diabetics, encodes susceptibility to disease. Analysis of the immune response to myelin basic protein (MBP) in mice, which leads to autoimmune experimental allergic encephalomyelitis, has shown that different MBP peptides can be recognised by encephalitogenic T cells that are

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restricted by different I-A and I-E alleles [128]. It is possible, therefore, as has been suggested previously [53], that different DQ molecules such as A3/DQw2 or A3/DQw4 or A3/DQw8 present different peptide epitopes of the same autoantigen. The efficiency of peptide presentation is proposed to be a critical factor in susceptibility. The alternative view is that DR4, DR3, DR2, DR8 and DR1 haplotypes contain susceptibility alleles at distinct loci throughout the MHC. Certainly, for DR8 and DR1 haplotypes there are no published data that map susceptibility convincingly to the DQ subregion. Whichever view is correct and, at this time, the weight of evidence favours a role for DQ alleles, non-sharing of haplotypes in families, particularly families with two or more affected siblings, may not reflect MHCsusceptibility determinant non-sharing. Therefore, calculations to assess the contribution of the MHC, based on the frequency of siblings that are concordant for disease but do not share either MHC haplotype may underestimate the contribution of the MHC [80]. Siblings that are non-DR haplotype sharing may share DQ or other determinants, such as "residue 57"-type polymorphisms, which may occur in association with several different alleles and have a significant effect on susceptibility in each case. The associations of DQ alleles with susceptibility to disease described above suggest that the contribution of the MHC may be considerable. Proof of roles for DQ molecules in susceptibility awaits identification of the T cell autoantigen and isolation of human T cell clones. T cell clones have been isolated from Type 1 diabetics and invariably are DR restricted [119]. Almost all human antigen-specific T cell clones isolated in different system, to date, have been DR restricted. This is probably because DR is expressed at high levels in peripheral blood lymphocytes, whereas DQ is hardly detectable (by fluorescent antibody labelling). That T cell clones from diabetics are DR restricted does not argue against a role for DQ-restricted T cells in disease.

15 DR and DQ polymorphisms for identification of high-risk individuals The PCR allows screening of MHC class II alleles in a large number of individuals. From what we know of the DQ and DR alleles that are associated with disease, it is possible to identify individuals in the population who are at an absolute risk of about 6% - 8 % [74, 98]. This figure is much better that that calculated from DR serological data: DR3/4 heterozygotes are at 3 % risk [107]. Improved identification of polymorphisms that are more closely associated with disease has led to a more accurate determination of risk (presumably attributable to the class II region). It is interesting to note that in a recent study DQw8-positive, DQw7negative DR4/4 homozygotes are at the same risk of disease as DR3/4 individuals [74]. An important goal of genetic research is to identify individuals who are at high risk of disease. Such individuals could be subjects for investigations into the appearance of autoantibodies or metabolic defects that may be associated with early events of disease pathogenesis or, possibly, be suitable participants in future clinical trails.


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Another area that requires further research concerns the consistent finding that the sex of the parent influences the risk of siblings [103, 111]. The risk to children is higher in families with an affected father compared to those with an affected mother. It has been shown that this effect is not due to transmission distortion at the MHC. One possible mechanism is that disease penetrance depends on the sex of the parent contributing the predisposing gene [26].

16 Analysis of non-MHC genes Many candidate genes, other than MHC genes have been tested for association with susceptibility to Type 1 diabetes. Linkage analysis of immunoglobulin allotypes and insulin gene polymorphisms were analysed recently and no evidence of cosegregation of these genes with disease was found [22, 26, 103]. For the insulin gene, this result conflicts with population association data [8, 45, 129]. It is possible to obtain an association of a marker in a population in the absence of haplotype in family studies if the disease allele occurs on a common haplotype [21, 112]. The insulin gene family data from the Genetic Analysis Workshop 5 were analysed in a different way [26, 112]. Disease-associated alleles were identified and the frequencies of these compared with those of insulin alleles that were not associated with disease in families. Application of this method confirmed the predisposing effect associated with the class I allele of the polymorphic region 5' to the insulin gene [112]. In most caucasian population studies, but not in different ethnic groups, an association of this polymorphism with susceptibility to Type 1 diabetes has been found [8, 45, 129]. An alternative explanation for the lack of insulin gene haplotype sharing in families was proposed: it could be that the effect of the insulin gene region is weak. Cox and Spielman [22] used computer simulation to estimate the contribution of the insulin gene that could give rise to a population association but fail to produce distorted segregation of haplotypes. They found that if the homozygous class I genotype at the insulin locus enhances the risk conferred by another locus (i.e the MHC) even as little as twofold, the frequency of class I alleles in Type 1 diabetics may be increased significantly without any apparent increase in class I allele haplotype sharing in 100 affected sib pairs. These data suggest that conventional linkage studies, even in large numbers of pedigrees, can have limited resolving power for identification of disease susceptibility alleles. Other approaches, such as the method used by Field [26] and Thomson [112] can yield valuable information. Nevertheless, the insulin gene or a gene that is closely linked to it, probably contributes to susceptibility to disease. A powerful strategy to map other human genes that influence susceptibility is to identify the NOD loci responsible for diabetes. At least three genes (and perhaps as many as six) determine beta cell destruction [35, 77, 122]. Two of these genes have been mapped, Idd-1 to the MHC [35, 77, 122] and Idd-2 to chromosome 9 [77]. Since the pathology and autoimmune features of the human and murine diseases are very similar and the class II defect appears to be conserved (Section 8), it is reasonable to predict that other non-MHC murine susceptibility genes may be conserved. Comparative mapping in mouse and human is becoming

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a extremely useful and productive strategy because it appears that the arrangement of many genes are conserved between the species [59]. Because large pedigrees can be established in NOD crosses with diabetes-resistant strains, the task of identifying susceptibility loci by linkage analysis is more feasible than the equivalent study in human families. It is also possible to fix certain features of the disease, such as insulitis, on resistant backgrounds to analyse the genetics of the defect and faciltitate mapping of the genes responsible [122]. Conversely, possible linkages found in the human can be analysed in mouse either to confirm linkage and/or facilitate fine mapping using mouse breeding to generate informative recombinants. A combined genetic and biochemical approach will greatly aid understanding of the mechanisms of beta cell destruction.

17 Genes and environment The high discordance of genetically identical individuals indicates that the environment is an important determinant of disease development. Disease prevalence in inbred, genetically identical female NOD mice is about 50 % - 90%, depending on the colony. The level of disease can be increased to almost 100% (in both males and females) by breeding NOD mice under germ-free conditions [ 106] or in certain cases, under specific pathogen-free conditions [86]. Immune stimulation by injection of adjuvant [86] or cytokines (e.g. IL-2; [96]) or viral infection [69] can prevent diabetes in the NOD mouse. It is possible that non-specific immune stimulation reduces the level of autoantigen presentation by class II molecules and even a minor decrease in class II-mediated susceptibility may be sufficient to prevent disease development. The viruses rubella, Coxsackie and CMV have been implicated as environmental triggering agents for Type 1 diabetes [75, 127]. The evidence for the associations of these viral infections with Type 1 diabetes is not conclusive and the search for a human diabetogenic virus continues. Recent analysis of Coxsackie B4 antibodies in Type 1 diabetic families showed that the prevalence of antibodies to the virus did not differ between affected and unaffected siblings [ 127]. It may be that any viral infection of beta cells in a genetically susceptible individual can trigger or stimulate beta cell destruction by the immune system. In Section 13 it was speculated that immune responsiveness to CMV is linked to susceptibility to Type 1 diabetes. Conversely, viral or bacterial infections may serve to protect against the development of disease. Sharing of infection may account for the high discordance of some identical twins. Perhaps improved healthcare and vaccination against common viruses has contributed to the increasing incidence of Type 1 diabetes observed in several countries.

18 Conclusions Allelic sequencing combined with comparative analyses of population-specific haplotypes have provided persuasive evidence that the Type 1 diabetes susceptibility determinants associated and linked to the MHC class II region are partly


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due to D Q allelic p o l y m o r p h i s m . A central role for an i m m u n e response m o l e c u l e in this disease is consistent with m a n y o f the features of T y p e 1 diabetes. Identification of autoantigens and c l o n i n g o f pathogenic T cells will help elucidate the roles o f class II alleles in disease. This and m a p p i n g o f other genes both inside and outside the M H C will eventually lead to a way o f p r e v e n t i n g or at least decreasing beta cell destruction.

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Erratum to: Feature ranking of type 1 diabetes susceptibility genes improves prediction of type 1 diabetes.

Genetic analysis of HLA class II alleles and susceptibility to type 1 (insulin-dependent) diabetes mellitus in Japanese subjects.

Genetic analysis of autoimmune type 1 diabetes mellitus in mice.

Feature ranking of type 1 diabetes susceptibility genes improves prediction of type 1 diabetes.

The Genetic Architecture of Type 1 Diabetes.

Genetic susceptibility in diabetes mellitus: analysis of the HLA association.

Genetic polymorphism of apolipoprotein A5 gene and susceptibility to type 2 diabetes mellitus: a meta-analysis of 15,137 subjects.

TNF-α and IFN-γ gene variation and genetic susceptibility to type 1 diabetes and its microangiopathic complications.

Association of vitamin D receptor polymorphisms and type 1 diabetes susceptibility in children: a meta-analysis.

Contribution of VDR polymorphisms to type 1 diabetes susceptibility: Systematic review of case-control studies and meta-analysis.

Genetic control of autoimmunity in type 1 diabetes.

Genetic analysis of a complex, multifactorial disease, autoimmune type 1 (insulin-dependent) diabetes.

Association of CD226 polymorphisms with the susceptibility to type 1 diabetes in Chinese children.

Elevation of plasma 1-deoxy-sphingolipids in type 2 diabetes mellitus: a susceptibility to neuropathy?

Personal history of diabetes, genetic susceptibility to diabetes, and risk of brain glioma: a pooled analysis of observational studies.

Genetic and Pharmacologic Models for Type 1 Diabetes.

T-cell receptor constant beta chain polymorphisms and susceptibility to type 1 diabetes.

Genome-wide trans-ancestry meta-analysis provides insight into the genetic architecture of type 2 diabetes susceptibility.

Host susceptibility factors to bacterial infections in type 2 diabetes.

Attitudes to Exercise and Diabetes in Young People with Type 1 Diabetes Mellitus: A Qualitative Analysis.

Untargeted metabolomic analysis in naturally occurring canine diabetes mellitus identifies similarities to human Type 1 Diabetes.

Role of Established Type 2 Diabetes-Susceptibility Genetic Variants in a High Prevalence American Indian Population.

Association of ACE and MTHFR genetic polymorphisms with type 2 diabetes mellitus: Susceptibility and complications.

Genetic analysis of the human type-1 angiotensin II receptor.