Islet autoantibodies appear long before the development of diabetes, and have proved invaluable in our understanding of the natural history of the disease. This section considers the role of autoantibodies in identifying individuals at increased risk of progression to diabetes, usually undertaken with a view to recruitment to intervention trials. The likelihood of progression to diabetes in autoantibody-positive individuals is influenced by baseline risk (genetic susceptibility), the age at which antibodies develop, the persistence and magnitude of the antibody response, the specificity of antibodies (i.e. the presence or absence of certain epitopes known to be associated with high risk), and by the number of antibody species that are present. Progression to diabetes is almost inevitable in those with multiple antibodies, and progression within 3–5 years is probable in those who also have evidence of target organ damage, i.e. impaired oral glucose tolerance or a reduced first-phase insulin response to intravenous glucose.
Role of antibodies in disease prediction
Much of our current understanding of islet autoantibodies and their role in disease prediction comes from prospective studies in individuals with an increased genetic susceptibility, such as first degree relatives or individuals with no family history who possess high risk HLA alleles. The prevalence of islet autoantibodies in relatives is 5–10%, depending upon which antibodies are measured. The Diabetes Prevention Trial Type 1 (DPT-1) screened samples from 17,207 first-degree relatives for ICA, GADA, IA-2A and IAA; the test was considered positive in those with antibody levels >99th percentile of a control population. 8.2% of affected relatives had at least one autoantibody, and 2.3% had more than one. Not all of those with islet autoantibodies will develop type 1 diabetes, and considerable effort has been made to identify disease-specific characteristics in order to distinguish between those who will and will not progress to diabetes, and the rate at which this might be expected to develop.
Age at autoantibody appearance
The earlier in life the first autoantibody appears, the greater the risk of progression of islet autoimmunity; the early appearance of multiple autoantibodies is associated with rapid progression to diabetes. In the German BABYDIAB cohort, 50% of children who developed multiple islet autoantibodies within the first year of life progressed to diabetes within 2 years of follow-up. This was significantly more rapid than 2-year progression in children who developed multiple islet autoantibodies at age 2 years (17%) or at age 5 years (7%). The magnitude of the autoantibody response in early childhood also appears to be greater than in later life. Children who develop islet autoantibodies before age 2 years frequently have high-affinity IAA and progress to multiple islet autoantibodies, whereas children who develop autoantibodies after age 2 years are less frequently IAA-positive, are often GADA-positive and infrequently develop multiple islet autoantibodies. Since age is strongly associated with antibody characteristics and genetic risk factors, it adds little to prediction models based upon islet autoantibodies alone. IAA are almost always the first autoantibodies to appear in young children who subsequently progress to diabetes, and the appearance of IAA is typically followed by rapid spread to other islet autoantibodies. Risk screening in children, adolescents and young adults (up to about 25 years of age) should therefore include all four major autoantibodies, i.e. IAA, GADA, IA-2A and ZnT8A.
The prevalence of IAA decreases dramatically with increasing age and IAA are therefore not particularly useful screening markers in the over-25-years age groups. The prevalence of GADA on the other hand, is relatively stable with age. IA-2A are slightly more prevalent in younger cases, whereas the prevalence of ZnT8A is directly correlated with the age of the onset of type 1 diabetes. Adults >25 years old should therefore be screened for GADA, IA-2A and ZnT8A, with additional sequential testing for IAA for further risk stratification.
Transient autoantibody appearance is not associated with progression to diabetes. IAA are least likely to persist among the major islet autoantibodies and this is related to antibody titre. In small children, transient IAA are also associated with maternal transfer of IAA.
The magnitude of an autoantibody response is reflected by persistence, titre, affinity and the breadth or range of autoantigen targets. Diabetes development has been associated with high titre ICA, IAA or IA-2A. High titre also determines other characteristics such as breadth of the response in terms of IgG subclass usage and epitope reactivity. As expected, high titre responses are usually synonymous with multiple IgG subclass antibodies to multiple epitopes, though these features can also be independent indicators of disease risk in low titre autoantibody-positive subjects. In an analysis of autoantibody-positive relatives followed for up to 15 years, the highest risks for type 1 diabetes were associated with high titre IAA and IA-2A responses, with the appearance of antibody subclasses IgG2, IgG3, and/or IgG4 of IAA and IA-2A, and antibodies to the IA-2-related molecule IA-2β. Using various combinations of these islet autoantibody characteristics it was possible to stratify 5-year diabetes risk from less than 10% to about 90%.
There appears to be a hierarchy of diabetes-relevance in the autoantibody response against different antigenic targets within and between islet autoantigens. For example, whereas risk is relatively low in relatives with GADA or IAA alone (about 20% within 10 years), the presence of IA-2A alone is associated with a similar risk (about 50% within 10 years) to multiple non-IA-2-autoantibodies (ICA, GADA and/or IAA). Among IA-2A positive relatives, risk can be further stratified according to the presence or absence of autoantibodies to IA-2β. In addition, IAA without proinsulin reactivity are associated with low risk, whereas proinsulin-reactive IAA are associated with very high risk of progression. For GADA, the N-terminal GAD-restricted antibodies are associated with low/no risk of progression, whereas individuals with antibodies directed towards the middle and/or C-terminal of the antigen progress to disease.
Antibody affinity provides a measure of maturity of the immune response. In a typical antibody response, exposure to antigen in the presence of B-cell growth factors results in B-lymphocyte expansion and IgM antibody production. Sustained or repeated antigen exposure leads to a switch from IgM to IgG production, and selection of clones that produce antibodies of high affinity to the antigen. In type 1 diabetes, high-affinity autoantibodies are associated with progression and are therefore 'diabetes-relevant', whereas low affinity antibodies are not. In BABYDIAB, IAA affinity varied considerably between IAA-positive children. Those who developed high-affinity IAA (Kd >109 l/mol) had persistent IAA, developed multiple islet autoantibodies and had a 50% risk of developing diabetes within 6 years. In contrast, children with IAA of lower affinity rarely progressed to multiple islet autoantibodies or diabetes. Furthermore, high-affinity IAA differed from lower affinity IAA in insulin binding characteristics suggesting distinct epitope recognition and, in contrast to the lower affinity IAA, the associated epitope was also expressed on the proinsulin molecule. Similar findings were obtained for GADA, in that single high-affinity GADA-positive children progressed to multiple islet autoantibodies and type 1 diabetes more frequently than children with low-affinity GADA.
Multiple islet autoantibodies
The development of multiple antibodies represents a critical step in pathogenesis, and is therefore a powerful marker of risk of progression. Identification of two or more islet autoantibodies is associated with a significantly greater risk than a single autoantibody. Whereas type 1 diabetes risk is less than 20% in relatives with just one islet autoantibody, it is about 35% within 5 years and 61% within 10 years in those with more than one autoantibody. Individuals with multiple islet autoantibodies but no family history of diabetes also appear to be at high risk.
The characterisation of an individual as 'multiple autoantibody-positive' does, however, depend on which markers are tested. For example, about two-thirds of relatives found to be GADA positive, but IAA and IA-2A negative (i.e. previously defined as low risk) in fact had autoantibodies to the newly identified autoantigen ZnT8, moving them into the multiple antibody-positive category. It is therefore possible that further marker(s) will become available that can identify more advanced islet autoimmunity and higher risk among individuals who are currently categorised as 'single autoantibody-positive'.
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