Type 1 Diabetes. Åke Lernmark. Clinical Chemistry 45:8(B) (1999) Beckman Conference

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1 Clinical Chemistry 45:8(B) (1999) Beckman Conference Type 1 Diabetes Åke Lernmark Type 1 (insulin-dependent) diabetes occurs worldwide and can appear at any age. The genetic susceptibility is strongly associated with HLA-DQ and DR on chromosome 6, but genetic factors on other chromosomes such as the insulin gene on chromosome 11 and the cytotoxic T-lymphocyte antigen gene on chromosome 2 may modulate disease risk. Numerous studies further support the view that environmental factors are important. Gestational infections may contribute to initiation, whereas later infections may accelerate islet -cell autoimmunity. The pathogenesis is strongly related to autoimmunity against the islet cells. Markers of autoimmunity include autoantibodies against glutamic acid decarboxylase, insulin, and islet cell antigen-2, a tyrosine phosphatase-like protein. Molecular techniques are used to establish reproducible and precise autoantibody assays, which have been subject to worldwide standardization. The diagnostic sensitivity (40 80%) and specificity (99%) of all three autoantibodies for type 1 diabetes are high, and double or triple positivity among first-degree relatives predicts disease. Combined genetic and antibody testing improved prediction in the general population despite the transient nature of these autoantibodies. Classification of diabetes has also been improved by autoantibody testing and may be used in type 2 diabetes to predict secondary failure and insulin requirement. Islet autoantibodies do not seem to be related to late complications but rather to metabolic control, perhaps because the presence of islet cell autoantibodies marks different residual -cell function. Combined genetic and autoantibody screening permit rational approaches to identify subjects for secondary and tertiary intervention trials American Association for Clinical Chemistry Diabetes mellitus is a heterogeneous group of disorders, all characterized by increased plasma glucose. In the majority of patients with diabetes, the etiology of the disease is not understood. Expert panels have recommended one set of criteria for diagnosis and another set for classification (1, 2). The criteria serve two purposes. One is to secure optimal treatment of the patient. The other is to support research aimed at understanding the etiology and pathogenesis of diabetes syndromes. Recently, new guidelines (Table 1) have been suggested for the concentrations of blood glucose to be used to diagnose diabetes (3). Normal fasting plasma glucose (FPG) 1 is 6.1 mmol/l (110 mg/dl). Impaired fasting glucose is 6.1 mmol/l (110 mg/dl) and 7.0 mmol/l (126 mg/ dl). Provisional diagnosis of diabetes is made at a FPG 7.0 mmol/l (126 mg/dl). It is recommended that the test is repeated on a different day for the final diagnosis of diabetes. Many recent investigations, therefore, support the view the diabetes mellitus syndrome is very heterogeneous. The most common type of diabetes is type 2 diabetes. The etiology is still unclear. The major recent advances in understanding the etiology of diabetes have come from genetic investigations of monogenic types of diabetes, most prominently the primary genetic diabetes classified as MODY 1 5 (4 6). Gestational diabetes is still a major problem affecting 4% of all pregnancies. Mothers with gestational diabetes have a markedly increased risk of developing postpartum diabetes. Type 1 diabetes is the most severe type of diabetes, leading to life-long dependency on daily insulin injections. In adults, type 1 may often masquerade as type 2 diabetes (7, 8). Diabetes is sufficiently common that it is possible that the genetic risk for one type may contribute to the risk of developing another type of diabetes. The importance of modifying factors is again exemplified by patients, often referred to as having latent autoimmune diabetes in the adult, who seem to have a slow-onset type 1 autoimmune diabetes. In the present short review, the current progress in understanding the etiology and pathogenesis of diabetes will be discussed along with recent evaluation of assays to determine diagnostic sensitivity and specificity as well as R.H. Williams Laboratory, University of Washington, 1959 N.E. Pacific St., Room K-165, HSB, Seattle, WA Fax ; ake@u. washington.edu. Received May 3, 1999; accepted May 27, Nonstandard abbreviations: FPG, fasting plasma glucose; CTLA-4, cytotoxic T-lymphocyte antigen-4; GAD65, glutamic acid decarboxylase; and IA-2, islet cell antigen

2 1332 Lernmark: Type 1 Diabetes Table 1. Diagnosis and classification of diabetes mellitus. A. Diagnosis FPG Diagnosis 6.1 mmol/l (110 mg/dl) Normal 6.1 mmol/l (110 mg/dl) Increased fasting glucose to 7.0 mmol/l (126 mg/dl) 7.0 mmol/l (126 mg/dl) Provisional diagnosis B. Classification Type 1 diabetes Idiopathic type 1 Type 2 diabetes Other specific types of diabetes (Primary genetic diabetes) Gestational diabetes mellitus the usefulness of combined genetic and antibody tests to predict type 1 diabetes. The possible role of autoimmunity in type 1 diabetes in relation to long-term complications will also be discussed briefly. The reader is referred to more extensive reviews of recent progress made in research to understand the development and possible future prevention of type 1 diabetes (4, 9 14). Etiology We are far from understanding the etiology of type 1 diabetes (Table 2). Several genetic factors have been identified that indicate the presence of strong susceptibility genetic factors (9, 15). It is well known that studies in identical twins demonstrate a concordance rate below 50%, supporting the view that although genes are important, environmental factors may be even more important. It has been known for the past 150 years that type 1 diabetes may develop in conjunction with certain viral infections (16). It is, therefore, often speculated that the combinations of susceptibility genes with environmental factors are important to the development of type 1 diabetes. Genetic Factors Many studies have shown that regardless of the ethnic background, type 1 diabetes is strongly genetically linked and associated with HLA on chromosome 6 (Table 2). Molecular cloning and sequencing of HLA genes and class II proteins have made it possible to study genetic loci that Table 2. Etiology Genetic susceptibility HLA DR3-DQ2, DQ8, or both Insulin CTLA-4 Many other genetic factors are candidates Environmental factors causing -cell destruction or release of autoantigens Gestational infections -cell toxins Other infections may explain the strong association and linkage to type 1 diabetes. Recent studies have indicated that HLA- DQB A is more important than any of the 12 or more HLA-DRB1 04 subtypes (17 19). However, to complicate the understanding of the role of HLA class II molecules in disease, it is also demonstrated that the HLA-DRB1 04 subtype, DRB1*0401 confers a risk that is additive to that of DQB A (20, 21). In other words, it may be argued that the DR*0401 allele confers a risk for diabetes that is independent of DQ. Similarly, on the HLA DQB A DRB1 03 extended haplotype, the DRB1 03 haplotype seems to provide an equal risk or slightly higher risk for type 1 diabetes than DQB A (22). Although molecular combinations in cis and trans may explain possible ways by which class II molecules are formed and are able to present antigen (23), further studies are warranted to explain the mechanisms of disease susceptibility. HLA-DQ6 is negatively associated with type 1 diabetes. It is speculated that the HLA DR or DQ class II molecules associated with type 1 diabetes provide antigen presentations that generate T-helper cells that initiate an immune response to specific islet cell autoantigens. This immune response includes the formation of cytotoxic T cells, which kill the insulinproducing cells in the islets of Langerhans, and also leads to the formation of autoantibodies. In the absence of reliable T-cell assays, the above possibility remains difficult to test. It has been estimated that 60% of the genetic susceptibility to type 1 diabetes is conferred by HLA (24). Several approaches to identify other susceptibility genes have therefore been taken. Families with multiple affected members have been collected (25) and used by several laboratories to carry out complete genome scans to identify other type 1 diabetes genes (26, 27). Currently, there are 15 such candidate loci identified. It is possible that a combination of HLA with other genetic factors may either enhance or decelerate the type 1 diabetes process. Some prominent candidate factors are the insulin gene on chromosome 11 (28, 29) and the cytotoxic T-lymphocyte antigen (CTLA-4) gene on chromosome 2 (30). Upstream of the insulin gene are variable numbers of tandem repeats (28). Class I alleles (26 63 repeats) predispose in a recessive way to type 1 diabetes, whereas class III alleles (140 to 200 repeats) seem dominantly protective. The protective effect may be explained by higher concentrations of proinsulin mrna in the thymus, perhaps to enhance immune tolerance to preproinsulin, a key autoantigen in type 1 diabetes pathogenesis (31, 32). The linkage to the CTLA4 gene is not understood; however, it has been speculated that a gene polymorphism involving a AT repeat at the C terminus at the 3 end of the gene may affect the mrna stability of CTLA-4 mrna. The longer the repeat, the less stable the CTLA-4 mrna. Because CTLA-4 is critical to T-cell apoptosis, it has been speculated that long AT repeats may lead to T-cell survival because the CTLA-4 protein is not formed. Further

3 Clinical Chemistry 45, No. 8(B), Country Virus antibody during pregnancy Table 3. Intrauterine exposure to enterovirus. a Child developing diabetes (n) b Controls (n) b P Sweden IgG CBV B (57) 0.32 (203) 0.02 Echo Echo IgM Echo Finland IgM CBV B (38) 0.03 (38) a From Dahlquist et al. (38) and Hyötyetal.(39). b Levels of antibodies are shown with the number of subjects in parentheses. experiments will be necessary to uncover the importance and role of other type 1 diabetes genes. Environmental Factors Studies in both dizygotic and monozygotic twins are consistent with the fact that the environment is important to the risk of type 1 diabetes (33, 34). Numerous casecontrol studies on the association of recent outbreaks of virus infection mumps, Coxsackie B, Echo virus, and the onset of type 1 diabetes have been published. The most compelling evidence is congenital rubella, which is strongly associated with appearance in the affected child (35 37). More recent studies have provided evidence that maternal enterovirus infections increase the risk for type 1 diabetes in the offspring (38, 39). Recently it has been demonstrated that intrauterine exposure to enterovirus (Table 3) is also associated with an increased risk of the offspring developing type 1 diabetes (38, 39). It has been speculated that a combination between susceptibility genes and environmental factors may initiate a disease process that is associated with a formation of an autoimmune response to the insulin-producing cells. Pathogenesis This autoimmune reaction is reflected by the presence of antibodies against prominent antigens in the pancreatic cell. The HLA type of the individual may control the recognition of certain autoantigens. The most important markers for -cell autoimmunity are autoantibodies against insulin (40, 41), glutamic acid decarboxylase (GAD65) (41, 42), and islet cell antigen-2 (IA-2) (41, 43, 44). All three molecules are available as recombinant molecules, which has made development of highly sensitive and reproducible assays for autoantibodies against these autoantigens possible (41). Radioligand binding assays using in vitro transcribed and translated GAD65 and IA-2 demonstrate high diagnostic sensitivity and specificity for type 1 diabetes (Table 4). Monoiodinated insulin is used in a radiobinding assay (45, 46). ELISA tests are not appropriate to detect these autoantibodies, which are shown to be dependent on conformational epitopes (47, 48). Diagnostic Sensitivity and Specificity The Immunology of Diabetes Workshop and Immunology of Diabetes Society have organized antibody standardization workshops since 1985 (49, 50). The recent Combinatorial Islet Autoantibody Workshop (41) demonstrated that GAD65Ab and IA-2Ab have a high diagnostic sensitivity and specificity for type 1 diabetes (Table 4) and can be measured consistently by most laboratories. The diagnostic sensitivity of insulin autoantibodies is age dependent and decreases with increasing age. The assay concordance for insulin autoantibodies in different laboratories was markedly less than for IA-2Ab and GAD65Ab, respectively. The Combinatorial Islet Autoantibody Workshop, however, demonstrated that the use of a combination of autoantibody assays made it possible for several laboratories to achieve excellent discrimination between diabetic and control sera (Table 5). Many laboratories achieved a sensitivity of up to 80% with a false-positive rate of 0%. This international workshop standardization effort demonstrated that a combination of three assays might be used not only to identify new onset patients with type 1 diabetes but also to define criteria for inclusion in immune intervention and other trials. Apart from selecting subjects for immune intervention trials, the islet cell autoantibody tests may find use in the clinical routine to better classify adult patients with diabetes. Diabetes Classification Islet cell autoantibody assays, in particular the quantitative radioligand binding assays for GAD65Ab and IA- 2Ab, have also been important to diabetes classification (Table 1). Many patients with type 2 diabetes are antibody positive (7, 8, 13, 51). It has been demonstrated that plasma C-peptide concentrations as a measure of residual -cell function are decreased in patients classified with type 2 diabetes but positive for GAD65Ab (7, 8, 52). As many as 10% of new-onset patients with diabetes but classified with type 2 diabetes may have either GAD65Ab or IA-2Ab. These patients have substantially less plasma C-peptide than the antibody-negative type 2 diabetes patients (52). Follow-up studies on patients with type 1 Table 4. Diagnostic sensitivity and specificity of autoantibodies for type 1 diabetes. Autoantigen Sensitivity, % Specificity, % Insulin GAD IA

4 1334 Lernmark: Type 1 Diabetes Table 5. Combinatorial Islet Autoantibody Workshop. a Autoantigen Assay b sensitivity, % Diagnostic Insulin RIA (22) 0 50 ELISA (2) 4 and 14 GAD65 RIA (33) ELISA (4) IA-2 RIA (19) ELISA (2) 5 and 38 a From Verge et al. (41). b Numbers of participating laboratories are shown in parentheses. diabetes in relation to C-peptide concentrations have also demonstrated that fewer younger individuals have residual -cell function at follow-up compared with patients who are older than 15 years of age when diagnosed and classified with type 1 diabetes. These observations support the notion that some individuals develop a milder form of type 1 diabetes that is reflected by the presence of autoantibodies and clinical classification in type 2 diabetes (13, 53). It was therefore tested whether GAD65Ab was related to an abnormal glucose tolerance test in healthy adult subjects (54). Approximately 1% and 0.8% of subjects exceeded the 99th percentile of GAD65Ab and IA-2Ab, respectively. In individuals with diabetic oral glucose tolerance test results, GAD65Ab and IA-2Ab were significantly higher (about threefold) than in individuals with normal oral glucose tolerance test results. GAD65Ab and IA-2Ab are, therefore, associated with impaired or diabetic glucose tolerance in the adult population. This observation together with the association between GAD65Ab concentrations and body mass index may indicate a possible relationship between islet autoimmunity and -cell function abnormalities with obesity and insulin resistance (54). Studies are therefore warranted to determine the predictive value of these islet cell autoantibodies as a predictor for the appearance of diabetes in the healthy population. Prediction Several studies have been carried out in first-degree relatives of type 1 diabetic patients (55). It should be noted that these studies may not be fully representative of type 1 diabetes in the general population because numerous epidemiological studies have demonstrated that only 10% of new-onset type 1 diabetes children have a firstdegree relative with the disease (10, 56). However, it has not been possible to screen for islet cell antibodies in the general population until recently. The many family studies, which currently are being evaluated, are difficult to interpret because the subjects were identified in follow-up studies that used the indirect immunofluorescence test for islet cell antibodies (55, 57). It recently has been shown, however, that the combination the three autoantibody assays seems to be sufficient to replace the immunofluorescence assay (58, 59). Several studies have shown that the presence of three antibodies predicts diabetes (60, 61). The presence of any one of the autoantibodies alone may not be predictive of disease; hence, long-term family studies have identified GAD65Ab-positive subjects who have remained normoglycemic, although some of these individuals have reduced -cell function (62). It is possible that these individuals have a very high sensitivity to insulin, which precludes them from developing hyperglycemia. The data suggest, however, that there may be other factors in marker-positive first-degree relatives that decelerate the pathogenetic process despite a major loss of cells. It is therefore clear that antibodies alone do not have a high predictive value for type 1 diabetes. Current research is focused on the possibility of combining genetic markers with antibody testing (63). The genetic markers DBQ A and DQB A are both often used as inclusion criteria, whereas the presence of HLA DQ6 is used as an exclusion criterion for participation in follow-up studies with or without intervention treatment. The possible utility of other genetic factors is yet to be determined. Intervention Type 1 diabetes is approached by primary, secondary, and tertiary intervention (Table 6). Primary intervention would include a treatment of all individuals. The possibility of using autoantigens as a type of vaccination is currently being explored not only in animal experiments but also in human tests. The selection of children on the basis of HLA type is being used to treat newborns with either oral or nasal insulin (11). Animal experiments have shown that treatment of spontaneously diabetic nonobese diabetic mice with GAD as a peptide, protein, or expressed in potatoes reduced diabetes (64, 65). Vaccination studies of children and adults remain a future possibility to test whether type 1 diabetes can be prevented. Secondary intervention (Table 6) involves screening for genetic, autoantibody, and other possible markers at birth, in school children, or in adults (61). Individuals classified with type 2 diabetes but positive for islet autoantibodies (representing slow-onset type 1 diabetes, latent autoimmune diabetes in the adult, or type 1.5 diabetes) are also being tested to determine whether they are suitable for immune intervention to preserve their -cell function. Recent studies in GAD65Ab-positive patients in Japan suggested that early insulin treatment preserves -cell function (66). Several intervention trials are pending, including the use of subcutaneous or oral insulin in the Diabetes Prevention Trial for Type 1 Diabetes (67), milk formula or nasal insulin in Finland (11), aerosol insulin in Melbourne (68), or nicotinamide in the European Nicotinamide Diabetes Intervention Trial (69). In the next few years we will learn important lessons concerning to what extent such intervention trials preserve -cell function in subjects at risk for type 1 diabetes. Finally, tertiary intervention (Table 6) involves the

5 Clinical Chemistry 45, No. 8(B), Table 6. Possible interventions in type 1 diabetes. Primary intervention Aim: induce tolerance to autoimmunity Vaccination before the appearance of autoreactive T cells and autoantibodies Strategy: oral vaccination Mother Child Oral insulin GAD potatoes Cholera toxin insulin Cholera toxin GAD Vaccination Children Adults Secondary intervention Screening for immune and genetic markers At birth School children Adults Type 2 diabetes Slow-onset type 1 diabetes Latent autoimmune diabetes in the adult Type 1.5 diabetes Type 3 diabetes Insulin intervention Immune intervention Tertiary intervention All type 1 diabetes patients to be treated (antigen-specific immune suppression) at onset or later Pancreas transplantation Islet transplantation Recurrence of disease is a problem treatment of newly diagnosed patients with type 1 diabetes. Previous studies have demonstrated that immunosuppression with cyclosporin and other agents has not been able to stop the pathogenetic process in new-onset patients (14). A future novel treatment is envisioned that represents an antigen-specific immune intervention. Animal experiments have demonstrated that administration of antigen, be it GAD65 or insulin, at the time of clinical onset may slow the disease process (12, 70, 71). Pancreas and islet transplantation is being used in attempts to replace insulin production in patients. Until recently, patients have mostly been selected for pancreas as well as islet transplantation after a prior kidney transplantation. Islet transplantation has not yet been very successful, although recent studies with highly purified human islets show prolonged function (72, 73). Allograft rejection despite long-term immunosuppression as well as recurrence of diseases (73) often explains the failure. In one study, the islet graft-specific cellular auto- and alloreactivity in peripheral blood from patients with failing islet allografts were compared to the reactivities in patients with functional grafts. The patients who remained C-peptide-positive for 1 year exhibited no signs of Table 7. Future control of type 1 diabetes. DCCT a lesson Implementation Prevention of complications DPT-1 lesson Early insulin treatment -cell rest Immune alleviation a DCCT, Diabetes Control and Complications Trial; DPT-1, Diabetes Prevention Trial for Type 1 Diabetes. alloreactivity, and their autoreactivity to islet autoantigens was only marginally increased. In contrast, rapid failure was accompanied by increases in graft-specific alloreactive T cells and sometimes in autoreactivity to islet autoantigens. T-cell reactivities in peripheral blood may therefore also be important influences on the survival of -cell allografts (74). Long-Term Complications Retinopathy, nephropathy, and neuropathy are the three major long-term complications of type 1 diabetes. Currently, no relationship has been observed between type 1 immune markers and development of nephropathy. Retinopathy on the other hand has shown increased risk among DQB A and DBQ A individuals and a inverse correlation to GAD65 antibodies (75, 76). Neuropathy is highly controversial. It was reported that GAD65Ab may be present in type 1 diabetes patients with neuropathy (77), but numerous subsequent studies have failed to confirm this early demonstration (78 80). Future Control of Type 1 Diabetes The Diabetes Control and Complications Trial lesson of diabetes control is a question of success of implementation (Table 7). It has been clearly demonstrated that good diabetes control is important for preventing or delaying the onset of late complications (81). A major effort is being made to implement the Diabetes Control and Complications Trial principles. Ongoing studies that involve subcutaneous or oral insulin treatment of individuals with -cell loss and with autoimmune markers should provide new information on the possibility of using insulin before the clinical onset of hyperglycemia (67). The mechanisms may be either -cell rest (82) or the possibility that the administration of insulin would alleviate the immune response in individuals who are not yet diabetic (Table 7). A total of 75 years of experience with insulin therapy in relatively large dosages at the time of clinical diagnosis have, however, failed to prolong the honeymoon period. The common view is, therefore, that antigen-specific immunosuppression will be needed in addition to insulin to halt the pathogenetic process.

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