The identification of islet cell antibodies (ICA) by immunofluorescence in 1974 was a key step in the recognition of type 1 diabetes as an immune-mediated disease. ICA recognise a composite of specific islet antigens, several of which have now been characterised at the molecular level. These include insulin autoantibodies (IAA), antibodies against the enzyme glutamic acid decarboxylase (GADA), the protein tyrosine phosphatase-related molecules IA-2A and IA2β, and the cation transporter ZnT8. Simple and specific tests for antibodies directed against these molecules have been developed and refined in the course of a series of international workshops. Islet antibodies can be used to study the natural history of pre-type 1 diabetes, to identify individuals at increased risk of diabetes development, to select high risk individuals for trials of immune intervention. They are also used to identify an immune pathogenesis in a subgroup of older patients with apparent type 2 diabetes. This is known as latent autoimmune diabetes in adults (LADA).
The identification of islet cell antibodies (ICA) was a key step towards recognition of type 1 diabetes as an immune mediated disease. Furthermore, the observation that ICA preceded the development of diabetes in high-risk relatives led to understanding that type 1 diabetes is not a disease of acute onset, as previously thought, but is preceded by a prodrome characterised by the appearance of autoantibodies in the circulation and progressing to evidence of declining beta cell function. This prodrome can be identified in early childhood and typically precedes diabetes onset by many years. This observation has opened the door to the possibility of analysis of environmental factors in the causation of the disease, of disease prediction, and of interventions intended to delay or prevent clinical onset. A further application has been the recognition that islet autoantibodies, particularly GADA, are present in a subgroup of older people presenting with apparent type 2 diabetes. GAD positive individuals are more likely to require insulin treatment, and the condition is known as LADA.
Circulating islet autoantibodies are present in sera from ~95% of children with new-onset diabetes, and provide evidence of an active and disease-specific B lymphocyte response. Standardisation of methods has allowed them to be reproducibly detected. Prospective studies have shown that autoantibody profiles differ between individuals and vary over time. The best-validated and most widely-used predictive markers are autoantibodies directed against the biochemically defined target antigens insulin (IAA), GAD65 (GADA), IA-2 (IA-2A) and ZnT8 (ZnT8A). Multiple antibodies are typically present in those who progress to diabetes, and are of particular value for disease prediction. Autoantibodies directed against a range of other autoantigens have also been reported, but their clinical utility and relevance to the pathogenesis of type 1 diabetes has not been confirmed, either because appropriate studies have yet to be performed or because the target molecule has not been identified and they are therefore difficult to measure accurately.
Islet cell autoantibodies (ICA)
ICA were first described in patients with type 1 diabetes and multiple endocrine deficiencies associated with organ-specific autoimmunity, and were subsequently identified in new-onset cases and in individuals at high risk of developing diabetes. ICA are measured by immunofluorescent techniques which identify any antibody that binds to human islet tissue in a non-specific manner. They were difficult to standardise because the assay is operator-dependent, varies according to the quality of the human pancreatic tissue used as a substrate, and recognises heterogeneous antibodies which vary between individuals. A series of workshop meetings resulted in reasonable standardisation of ICA and quantification in Juvenile Diabetes Foundation Units, but ICA testing has since been superseded by testing for specific autoantibodies against biochemically defined islet antigens.
Insulin autoantibodies (IAA)
Antibodies directed against injected insulin formed the basis of the first insulin radioimmunoassay, and were intensively investigated in the 1960s because of their possible interference with insulin action. It therefore came as a surprise when autoantibodies to insulin were described in insulin-naïve patients with new onset diabetes in 1983. Proinsulin was later recognised as another target, and there are distinct immunisation patterns with respect to the affinity and epitope specificities of these antibodies. IAA appear very early in the natural history of the type 1 prodrome, at about 1–2 years of age, and are usually the first islet autoantibody to be detected. They are especially common (>70%) in childhood diabetes, and are less frequently detected after adolescence. High-affinity IAA are generally more predictive of type 1 diabetes and are associated with appearance at a young age, association with HLA DRB1*04, subsequent progression to multiple autoantibody positivity, binding to human insulin A chain residues 8–13, and binding to proinsulin.
GAD autoantibodies (GADA)
The next major autoantigen to be identified in 1990 was a 65 kDa isoform of glutamate decarboxylase (GAD65). Autoantibodies to GAD (GADA) are found in >70% of people with type 1 diabetes at all ages and they are the characteristic marker of autoimmune diabetes in adults. GAD is also present in nervous tissue, and GADA can also be detected in certain neurological disorders and in conditions unrelated to diabetes. Critical epitope clusters that appear early in the GADA response have been identified, and the affinity and epitope specificity of the antibody response predicts progression in the disease process. In the early stages of diabetes-associated GAD65 autoimmunity, the epitopes recognised by GADA are predominantly located in the middle region of the protein, but at later stages they may extend to regions at the N-terminus. GADA are particularly important as markers of an immune pathogenesis in LADA, a condition identified in older people who present with apparent type 2 diabetes.
IA-2 and IA-2β autoantibodies
Two tryptic digest fragments of islet antigens from patients with type 1 diabetes were characterized in 1995. One, a 40 kDa fragment from the intracellular portion of a tyrosine phosphatase-like protein (PTPRN gene), is now referred to as IA-2ic or ICA512ic. Autoantibodies to IA-2 (IA-2A) are almost always detected together with other islet autoantibodies and are very specific for type 1 diabetes. The other 37 kDa tryptic fragment was identified as the IA-2-related protein IA-2β or phogrin. Since almost all autoantibodies that react with IA-2β also react with IA-2, clinical laboratories typically do not use IA-2β autoantibodies as a first line test, but these may be of particular value for identifying individuals at high risk of disease progression. Critical epitope regions/residues for IA-2A and IA-2β antibodies have been defined, and their hierarchy of risk for future type 1 diabetes has been described. Sub-reactivity to the IA-2β protein is strongly associated with progression to diabetes within 5 years.
The most recent major islet autoantigen, zinc transporter-8 (ZnT8), was discovered in 2007 by screening for highly expressed, islet beta-cell specific molecules. ZnT8 belongs to a large family of zinc transporters and is associated with the membrane of secretory granules of islet beta cells; the zinc within these granules forms a complex with insulin to develop storage crystals. Autoantibodies to ZnT8 (ZnT8A) are found in about 70% of patients and improve disease prediction. A principal epitope targeted by ZnT8A is influenced by the single amino acid at position 325 encoded as arginine, tryptophan or glutamine by different polymorphic variants of the ZnT8-encoding gene SLC30A8. Autoimmunity directed against the COOH-terminal region of ZnT8 is of particular prognostic significance, and ZnT8A-positive children who are homozygous for either arginine or tryptophan at position 325 are at greatest risk of disease progression.
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