GAD antibodies

Autoantibodies to Glutamate decarboxylase (GADA) have become the most commonly used predictive marker for type 1 diabetes. They were first associated with type 1 diabetes in 1990 through comparison of sera from patients with type 1 diabetes and stiff person syndrome. After IAA, GADA are the antibodies detected most often at seroconversion in infancy, with an incidence exceeding IAA in later childhood. They also persist into adulthood and act as a marker for adult-onset autoimmune diabetes. Most studies have indicated that GADA are more persistent in long-standing diabetes patients than IA-2A or ZnT8A. Glutamate decarboxylase (GAD) is found in neurons and the pancreas, where it is involved in the synthesis of gamma-aminobutyric acid (GABA).

Discovery of GADA

Antibodies to unknown antigens found in the islets were originally measured by islet cell cytoplasmic antibody (ICA) staining[1]. Over time the specific autoantibodies contributing to ICA were characterised, including GADA, which were originally identified through immunoprecipitation of a 64kDa protein from 35S methionine labelled rat islets by sera from patients with type 1 diabetes. Autoantibodies to GAD are detected in up to 80% of patients and in the majority of sera from individuals who went on to develop the disease [2]. The identity of this 64kDa protein was established through the critical analysis of patients with a rare disorder, stiff person syndrome. These individuals have antibodies that recognised both GABA-secreting neurones and islet cells. The major autoantigen in these patients had already been identified as GAD by the 1980s. Subsequently, Baekkeskov and colleagues were able to show in 1990 that the 64kDa protein, important in type 1 diabetes, was also GAD [3].

Function of GAD

The enzyme glutamate decarboxylase (GAD), with its cofactor, pyridoxal 5’phosphate (PLP), catalyses the α-decarboxylation of L-glutamic acid to synthesise the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in the pancreas. GABA regulates the function of β cells via paracrine and autocrine signalling [4]. There are two distinct isoforms of GAD: GAD67 and GAD65, encoded by genes on chromosomes 2q31 and 10p11, respectively. The GAD65 sequence is 76% homologous and 85% similar to that of GAD67 through the last 12 exons (residues 174-585). GAD67 is soluble, and mainly present in the cell body of neurons. GAD65, however, is more hydrophobic and is reversibly anchored to synaptic vesicle membranes of neurons and microvesicles in β-cells. GAD65 is the major autoantigen in type 1 diabetes, while GAD67 has low immunogenicity perhaps due to the greater flexibility of the c-terminal region [2][5]. GAD is localised to synaptic-like vesicles and to the cytosol, which is in contrast to the other major targets of humoral autoimmunity in type 1 diabetes, whose cellular localisation is within the insulin secretory granules.

GADA assay development

Autoantibodies to GAD were first measured by radioimmunoassay (RIA) in 1994 [6], and, over 20 years later, this remains the de facto gold standard method. Recombinant 35S methionine-labelled GAD produced by in vitro reticulocyte lysate transcription/translation is incubated with sera, resulting in immunocomplexes which are precipitated with protein A sepharose, washed and counted in a beta scintillation counter. The level of GADA in the sera is directly related to the amount of radiolabelled GAD bound.

Alternative techniques for measuring GADA have also been developed. These include ELISA [7], luciferase-based immunoprecipitation system (LIPS) [8] and, more recently, electrochemiluminescence (ECL) [9]. To improve and standardise measurement of islet autoantibodies (including GADA), international workshops and proficiency programmes have been run. For example, the Islet Autoantibody Standardization Program (IASP) (née Diabetes Antibody Standardization Program (DASP)) use blinded sets of control and patient sera to assess and improve comparability of GADA, IA-2A, IAA and ZnT8A measurements between laboratories [10][11]. A harmonized method was also published in 2009 using common assay protocols and thresholds for measurement of GADA and IA-2A for National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) consortia studies [12].

Analysis stemming from DASP and IASP GADA workshops highlighted differences in GADA levels in control sera, which were dependent on assay type, with ELISA, ECL and LIPS methods often showing improved specificity compared with RIA. Control sera, which gave a positive result by RIA but were negative by ELISA, were characterised and shown to bind exclusively to the N-terminus of GAD. It is widely recognised that antibodies specific only to the N-terminal domain of GAD65 have little association with progression to type 1 diabetes. Reactivity of sera from individuals who have gone on to develop type 1 diabetes has been shown to typically spread from the C-terminal and middle (PLP) region and finally to the N-terminal domains of the molecule[13]. These observations lead to the development of an N-terminally truncated GAD radiolabel from which the first 95 amino acids have been deleted: 35S-GAD65 (96-585). This truncated radiolabel may improve the performance of GADA RIAs [14], since autoantibodies measured with this construct were found to be more closely associated with diabetes risk in relatives of patients with type 1 diabetes [15]. Despite the potential for assay improvement, assays using truncated GAD have yet to be more widely adopted.

Significance of GADA in the development of diabetes

A wide range of genetic and environmental factors contribute to the pathogenesis of type 1 diabetes and the time taken for someone to develop hyperglycaemia, after autoantibodies are first detected, varies from months to decades. This is highlighted by the potential presence of different “primary” antigens, i.e. those detected first in people who progress to type 1 diabetes. Autoantibodies to GADA can be detected in children as young as 3 months old, but their appearance is more common above the age of 2 years. In approximately half of children screened from birth for the TEDDY study, GADA were the first autoantibodies detected either alone or in combination with IAA. In children who initially develop IAA, subsequent appearance of GADA identifies those at high risk of progressing to type 1 diabetes. In contrast to IAA, the prevalence of GADA at the onset of type 1 diabetes in children correlates positively with age and patients who present with disease in adulthood frequently have GADA. High titres of GADA and development of GADA prior to IAA are associated with the high diabetes risk HLA haplotype DR3-DQ2 [16]. The question as to whether these observations arise from different underlying aetiologies is being investigated by ongoing prospective studies.

Role in prediction of type 1 diabetes

Approximately 80% of patients diagnosed with type 1 diabetes in childhood are positive for GADA. Measurement of GADA is widely used as a preliminary screen for islet autoimmunity, requiring less sera and time than IAA measurement. They are an excellent early marker, found at high prevalence in all age groups of individuals who develop diabetes. The gold standard method for prediction of type 1 diabetes is measurement of a combination of GADA with IAA, IA-2A, and ZnT8A. Measurement of GADA and either IA-2A or ZnT8A, however, can provide a relatively specific and sensitive determinant of risk. It is widely recognised that multiple autoantibody positive individuals have a higher risk of developing type 1 diabetes within 5 years compared with single antibody positive individuals [17]. Nevertheless, individuals with high levels of GADA alone can progress to multiple islet autoimmunity and subsequent diabetes [18].

Role in autoimmune diabetes in adults.

Autoantibodies to GAD also provide one of the critical definitions of latent autoimmune diabetes in adults (LADA). The definition of this disease has not been formalised, but criteria suggested by the Immunology of Diabetes Society include patients over 30 years old at diagnosis, positive for at least one islet autoantibody, and not treated with insulin within the first 6 months after diagnosis. These patients often, therefore, share some characteristics of both type 1 and type 2 diabetes and could account for between 2-10% of patients currently diagnosed with type 2 diabetes. GADA are the most prevalent autoantibody detected, although ZnT8A may also be useful in defining these patients [2].

Role of GAD immunotherapy

Despite the wide prevalence of non-specific immune therapies in type 1 diabetes, the induction of tolerance specific to islet autoantigens is still the most attractive approach to disease prevention. The observation that GAD is an early target for the immune system during the development of type 1 diabetes makes it a good candidate for antigen specific immune therapy. In the most widely used animal model of type 1 diabetes, the non-obese diabetic (NOD) mouse, autoimmune loss of the beta cells can be prevented by injection with exogenous GAD65 peptides before the onset of insulitis [19]. These findings have led to a series of clinical trials testing whether subcutaneous administration of GAD65 formulated with aluminium hydroxide (GAD-alum) shortly after diagnosis with diabetes could slow the decline in insulin production. Unfortunately, despite promise in phase I and II trials, including evidence that GAD-alum induced humoral and cellular immune responses [20], the phase III trial showed no benefit from therapy [21]. As yet, no trials have investigated GAD immunotherapy in at risk populations before diabetes onset, but studies of different routes of administration and potential adjuvants for GAD therapies continue in newly diagnosed patients (https://clinicaltrials.gov/ct2/results?term=GAD-Alum&pg=1).

The presence of GADA at early stages in the immune response against the pancreas, and their persistence during progression to and after disease diagnosis, make them an invaluable tool for predicting and defining diabetes. Although testing for multiple islet autoantibodies improves the accuracy with which type 1 diabetes can be predicted, development of more disease-specific GADA assays will allow better targeting of therapeutic interventions towards those most likely to benefit. Furthermore, improved understanding of the contribution of these autoantibodies to disease progression may suggest new ways for preventing type 1 diabetes.

References

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  3. ^ Baekkeskov S et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic-acid decarboxylase. Nature. 1990;347(6289):151-6

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