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Keywords:

  • autoantibody epitopes;
  • autoimmune;
  • GAD65Ab;
  • LADA

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We analysed the beta cell-specific autoimmunity reflected in autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) in the prediabetic period of GAD65Ab-positive healthy adults who developed Type 2 diabetes (T2D) during a follow-up period of 10 years. We found that of the adults that tested GAD65Ab-positive at baseline (n = 25), six developed T2D and one developed Type 1 diabetes (T1D). Of the subjects that tested GAD65Ab-negative at baseline (n = 2209), 81 developed T2D, one developed T1D and four developed unclassified diabetes, indicating that the risk for GAD65Ab-positive healthy adults to develop diabetes is increased sixfold. The GAD65Ab epitopes were characterized in a competition radioligand binding assay using recombinant Fab derived of GAD65-specific monoclonal antibodies. We observed that the GAD65Ab epitope specificities in the prediabetic period changed dynamically. Specifically, the binding to a middle and a C-terminal epitope increased during the follow-up period (P = 0·03), causing a significant increase in the number of epitopes recognized (P = 0·03). These findings are similar to previous observations of dynamic changes in the prediabetic period of schoolchildren at high risk for T1D development. However, the character of the epitopes differs between the two populations, suggesting differences in the beta cell-specific autoimmune response in the prediabetic period of patients with latent autoimmune diabetes in adults (LADA) and T1D.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Markers for the autoimmune response to the pancreatic beta cells can be found years before the onset of autoimmune diabetes [1,2]. While some of these markers − autoantibodies directed to insulin (IAA) and insulinoma antigen 2 (IA-2Ab) − show an inverse correlation with age at onset, others, specifically autoantibodies directed to the smaller isoform of glutamate decarboxylase (GAD65Ab), show no and in some studies even a positive correlation with age at onset and are therefore particularly attractive markers for autoimmune diabetes in the adult population [3]. GAD65Ab-positive Type 2 diabetes (T2D) patients are found at 10% among the T2D population [4–7]. These patients often progress towards insulin dependency within the first 5 years of diabetes [8] and are often referred to as patients with latent autoimmune diabetes in adults (LADA) [6].

While the humoral autoimmunity of LADA patients at clinical diagnosis of disease has been described by us [9–11] and others [12–15], little is known about the prediabetic period of these patients. When the autoimmune process starts and what causes some GAD65Ab-positive subjects to progress to autoimmune diabetes, while others do not, remains unclear.

In recent studies we have identified GAD65Ab epitopes specific for Type 1 diabetes (T1D) [11,16]. The study of GAD65Ab epitopes may therefore be useful in the identification of the underlying autoimmunity. Moreover, we showed dynamic changes in the GAD65Ab epitope recognition in a group of healthy schoolchildren at high risk for the development of T1D [17]. However, the humoral autoimmune response preceding the development of LADA remains largely overlooked. Therefore we analysed the beta cell-specific autoimmunity reflected in GAD65Ab in the prediabetic period of GAD65Ab-positive healthy adults who developed T2D during a follow-up period of 10 years.

Subjects, materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Subjects

Our study population consisted of 2314 subjects who participated in a health programme in the county of Västerbotten, Sweden (VIP) between 1988 and 1992. The subjects were invited to VIP without any exclusion criteria and were aged 30, 40, 50 or 60 years [mean age 44·6 years, standard deviation (s.d.) 10·6] at baseline. There was an equal distribution between men and women. The subjects were all living in the community of Lycksele in Västerbotten County at baseline and represented 74% of the eligible population [18].

In 2003 a follow-up study on the cohort was conducted, when the incidence of World Health Organization (WHO)-defined diabetes was studied [19]. Of the initial 2314 subjects, 2234 (96·5%) were included. The incidence of diabetes was studied by linking the original participating subjects to the county register of diabetes patients, medical records in primary health care and hospital records. The death certificates and medical records of deceased subjects (n = 101) were analysed to identify a diagnosis of diabetes. Also, in 1998–2003, 1633 of 2234 (73%) subjects participated a second time in VIP. Five hundred and twenty-five subjects who did not participate in the re-examination were sent a questionnaire to report if they had diabetes. Five hundred subjects answered the questionnaire and gave permission for the review of their medical records. Those who had developed diabetes allowed us to contact their physician to verify the diagnosis and also donated a fasting blood sample, which was stored at the medical biobank as described below. The clinicians classified the type of diabetes and the treatment at follow-up was registered.

The subjects gave written consent and the Ethics Committee of the Medical Faculty, Umeå University, Sweden, approved the study.

Procedures

Both at the baseline visit between 1988 and 1992 and at the re-examination 10 years later in 1998–2003, the subjects were asked to perform an oral glucose tolerance test (OGTT) where capillary fasting capillary plasma glucose (fP-glucose) and 2h-plasma glucose (2hP-glucose) was analysed after fasting overnight. In our analysis the WHO recommendation for classification of OGTT results was used [19] and all subjects included in the study had either normal glucose tolerance (NGT; fPG < 7·0 mmol/l and 2hPG < 8·9 mmol/l) or impaired glucose tolerance (IGT; fPG < 7·0 mmol/l and 2hPG 8·9–12·1 mmol/l), i.e. all participants were non-diabetic. All participants were asked to donate blood samples for research. The donated blood samples were kept frozen at −80°C at the medical biobank at Umeå University Hospital.

Radioligand binding assays (RBA)

Plasma samples were analysed for GAD65Ab using a RBA [20,21]. Levels of GAD65Ab were expressed as a GAD65Ab index to correct for interassay variation according to the following formula: GAD65Ab index = (counts per minute (cpm) of the unknown sample − average cpm of three negative standards)/(cpm of the positive standard − average of three negative standards). The intra-assay coefficient of variation for duplicate determinations was 8·2%. At baseline a cut-off for GAD65Ab positivity was set at a GAD65Ab index of 0·17, which represented the 99th percentile among subjects with NGT at baseline. The plasma samples were also analysed for IA-2Ab in a RBA identical to the GAD65Ab RBA but using 35S-labelled IA-2 as a tracer [22]. A cut-off for IA-2Ab positivity was set at an IA-2Ab index of 0·03, which represented the 99th percentile among subjects with NGT at baseline.

At follow-up plasma samples were collected from the subjects who had developed diabetes. These samples were analysed for presence of GAD65Ab and IA-2Ab. Plasma samples were available in 80 of the 93 subjects who had developed diabetes. GAD65Ab were analysed by RBA as described above. The cut-off was defined as the 99th percentile among blood donors (32 U/ml). IA-2Ab were analysed with a commercial kit (RSR Ltd, Cardiff, UK). The cut-off was ≥ 1·0 kU/l according to the manufacturer. In the First and Second International GAD Autoantibody Workshops, our GAD65Ab assay showed 100% and 82% sensitivity and 100% and 96% specificity, respectively.

Recombinant GAD65-specific Fab used in this study

Monoclonal antibodies DPA, DPC and DPD were derived from a patient with T1D [23]; they recognize epitopes located at amino acids 483–585, 195–412 and 96–173, respectively [24,25]. Monoclonal antibody b96·11 was derived similarly from a patient with autoimmune polyendocrine syndrome Type 1 (APS-1) [26], and recognizes conformational epitopes located at amino acid residues 308–365 [25,26]. Monoclonal antibody N-GAD65 mAb was raised in mice to amino acid residues 4–22 of human GAD65 [27]. rFab of these monoclonal antibodies were cloned as described [11].

Epitope-specific RBA (ES-RBA)

The capacity of the rFab to inhibit GAD65 binding by human serum GAD65Ab was tested in a competitive RBA using Protein A Sepharose (PAS) (Zymed Laboratories, Carlton, CA, USA) as the precipitating agent. Fab lack the CH2 domain of the Fc region and do not bind Protein A. Serum samples were first analysed at a final serum dilution of 1 : 25. If the GAD65Ab index exceeded 1, the sample was further diluted until the GAD65Ab index was < 1. The rFab concentration necessary to compete its intact MoAb was used in the competition assays as described [11,28,29]. The cut-off for specific competition was determined as > 20% by using as a negative control rFab D1·3, specific to an irrelevant target, anti-hen egg white lysozyme, at 5 µg/ml.

Statistical analyses

Binding of GAD65Ab to GAD65 in the presence of rFab was expressed as follows: (cpm of [35S]-GAD65 bound in the presence of rFab)/(cpm of [35S]-GAD65 bound in the absence of rFab) × 100. All samples were analysed in triplicate determinations and the average intra-assay coefficient of variation was 4% (range 0·2–9·0%). Positive and negative controls were included on each plate to correct for interassay variations. Differences in competition were tested using the Wilcoxon matched-pairs test. A P-value < 0·05 was considered significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Incidence of diabetes at follow-up

At follow-up in 2003, 93 of 2234 (4·2%) subjects (M/W 44/49) had developed diabetes. Diabetes developed after a mean time of 9·2 (±2·9) years after the baseline examination. Clinical parameters of these patients are presented in Table 1. The classification of diabetes according to the medical records showed that 87 patients had developed T2D, two patients developed T1D and in four patients the type of diabetes remained unclassified.

Table 1.  Characteristics of glutamate decarboxylase (GAD65Ab)-positive subjects by follow-up status.
 Developing diabetes (n = 7)Not developing diabetes (n = 18)
  1. Summary of clinical parameters of GAD65Ab-positive individuals. Missing values among participants not developing diabetes; body mass index (BMI) (n = 2), OGTT (oral glucose tolerance test) (n = 2), c-peptide (n = 6). There were no missing values among those who developed diabetes; n.a.: not available.

Gender (M/F)5/26/12
BMI at baseline30·5 ± 5·327·3 ± 4·5
Age at baseline (years)52·9 ± 9·544·4 ± 9·8
Age at diagnosis (years)63·9 ± 10·5 
IGT (%)570
fC-peptide at baseline (nmol/l)0·86 ± 0·410·56 ± 0·13
fC-peptide at follow-up (nmol/l)0·91 ± 0·65n.a.

Incidence of diabetes in GAD65Ab-positive individuals

In all, 28% (seven of 25) and 6% (one of 17) of the GAD65Ab-positive subjects and IA-2Ab-positive subjects, respectively, developed diabetes. Two of these subjects required insulin treatment at the time of clinical diagnosis and were therefore classified as T1D patients. The IA-2Ab-positive subject was also positive for GAD65Ab, whereas two other double autoantibody-positive subjects did not develop diabetes. None of the GAD65Ab-positive individuals that developed diabetes lost their GAD65Ab positivity (Fig. 1 upper panel, Fig. 2a). Six of the patients who developed T2D and were GAD65Ab-positive at follow-up tested negative for GAD65Ab at baseline (Fig. 1 lower panel).

image

Figure 1. Flow-chart demonstrating the development of diabetes among glutamate decarboxylase antibody (GAD65Ab)-positive (upper panel) and -negative (lower panel) individuals.

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image

Figure 2. Glutamate decarboxylase (GAD65Ab) at baseline and follow-up. GAD65Ab titres are shown in (a). Serum samples were analysed for GAD65Ab epitope binding using rFab DPC (b), DPA (c), b96·11 (d), DPD (e) and N-GAD65 mAb (f). Binding in the presence of competing rFab is presented as percentage relative to binding to GAD65 in the absence of competitor (set as 100%). The cut-off for unspecific binding is indicated at 80% by the dashed line. Individual subjects are presented by the same symbol throughout this figure. To allow clear identification of all symbols, overlapping or partially overlapping symbols are shown in parallel positions.

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GAD65Ab epitopes in relation to development of diabetes

We analysed the humoral immune response in the GAD65Ab-positive subjects who developed T2D during the follow-up, by studying their GAD65Ab epitope pattern. Subjects with a GAD65Ab index above 0·1 (n = 6) in both the baseline and the follow-up sample were analysed using competition experiments with five rFab; DPA (C-terminal epitope), b96·11 (middle epitope), DPC (middle epitope), DPD (N-terminal epitope) and N-GAD65 mAb (N-terminal epitope) (Fig. 2). Epitope patterns at the initial visit were compared with those at the follow-up visit. We found that at baseline the binding to GAD65 for most of the samples (five of six) was reduced significantly by rFab b96·11. At follow-up, binding to GAD65 for most of the samples was reduced significantly by rFab DPC (four of six).

The median binding to GAD65 in the presence of rFab DPC and DPA were reduced significantly in the follow-up sample compared to the baseline sample (98–78% and 90–78%, respectively) (P = 0·03 for both rFab). Binding to GAD65 in the presence of rFab DPD and N-GAD65 mAb remained stable over the follow-up period (Fig. 2e,f). Binding to GAD65 in the presence of rFab b96·11 (Fig. 2d) showed an increase over the follow-up period for two individuals, while three individuals showed a decrease.

The median number of recognized epitopes increased from one epitope in the initial sample to 3·5 epitopes in the final sample (Fig. 3) (P = 0·03).

image

Figure 3. Epitope numbers recognized by glutamate decarboxylase (GAD65Ab)-positive adults that developed Type 2 diabetes during follow-up. Individuals are presented with the same symbols as in Fig. 2. To allow clear identification of all symbols, overlapping or partially overlapping symbols are shown in parallel positions.

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We also analysed the T1D patient who was GAD65Ab-positive both at baseline and at the follow-up visit (Fig. 4). This patient showed a decrease in binding to the DPC-defined epitope (45–86%) and in the number of epitopes recognized (3–0). This patient developed T1D 5 years before the follow-up analysis, therefore these changes may have occurred after the clinical onset of diabetes.

image

Figure 4. Glutamate decarboxylase (GAD65Ab) specificities and recognized epitope numbers by the GAD65Ab-positive adult who developed Type 1 diabetes during follow-up. GAD65Ab were analysed at baseline and at follow-up. GAD65Ab titres are shown in (a). Serum samples were analysed for GAD65Ab epitope binding (b) using rFab DPA (black square), b96·11 (black triangle), DPC (black diamond), DPD (black circle) and N-GAD65 mAb (white circle). Binding in the presence of competing rFab is presented as percentage relative to binding to GAD65 in the absence of competitor (set as 100%). Epitope numbers recognized by the individual at baseline and at follow-up (c).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We analysed the beta cell specific autoimmunity reflected in the GAD65Ab epitope specificities in a small group of initially healthy GAD65Ab-positive adults who developed T2D during follow-up. While these patients were classified as T2D patients by their physicians, they met the criteria for LADA patients, namely that they were GAD65Ab -positive and did not require insulin treatment at diagnosis. These GAD65Ab-positive healthy adults showed dynamic changes in their humoral immune response during the progression to diabetes. We found an overall increase in the number of recognized epitopes due partially to increased binding to two conformational epitopes, located at the C-terminus and the middle of the molecule.

In our study group 4·2% of the initial population developed diabetes; this is in agreement with previously reported incidence rates for Swedish populations [30]. While only six of the GAD65Ab-positive T2D patients had GAD65Ab titres high enough at both baseline and follow-up to allow a meaningful analysis, the strength of our study remains in the prospective longitudinal design of this population-based cohort, allowing us to detect rare events such as autoantibody positive subjects developing diabetes. Our study indicates that GAD65Ab-positive non-diabetic subjects have more than a sixfold increased risk of developing diabetes compared to GAD65Ab-negative subjects.

Our previous analysis of prediabetic schoolchildren suggested longitudinal maturation of GAD65Ab that may reflect changes in the autoimmunity leading to the development of the disease [17]. We observed the same trend in this study − namely, changes in epitope recognition over time and an increase in the number of epitopes recognized. However, while in prediabetic schoolchildren the binding to the DPD-defined N-terminal epitope and the b96·11-defined middle epitope increased over time, we found in the adult population an increased binding to the DPA-defined C-terminal epitope and the DPC-defined middle epitope. Our data are supported by previous studies identifying GAD65Ab specific to the C-terminus as a strong indication of subsequent insulin requirement in LADA patients [12,13]. These differences in intramolecular epitope spreading may suggest different intensities of the underlying autoimmunity in these two populations. Whether the intramolecular epitope spreading observed here is influenced by possible protective factors, leading to a prolonged prodomal period, remains to be analysed. We hypothesize that the analysed patients will continue towards insulin dependency. A longitudinal analysis of the patients until onset of insulin requirement will be essential for the study of the underlying autoimmunity. Our hypothesis, that these patients are at an early stage of their beta cell autoimmunity, is supported by the observation that the epitope pattern and the low number of recognized GAD65Ab epitopes were similar to those observed in GAD65Ab-positive healthy [16,17] and in first-degree relatives of patients of T1D patients [16].

In conclusion, GAD65Ab are associated with the development of future diabetes among adults in our population-based cohort study. These autoantibodies show dynamic changes as the individual progresses towards diabetes.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was supported by the National Institutes of Health (DK53456, DK53004, DK026190), the Västerbotten County Council, Umeå University, the Swedish Society of Medicine and an ADA Career Development Award to C. S. H.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Subjects, materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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