Autoantibodies to N-terminally truncated GAD improve clinical phenotyping of individuals with adult-onset diabetes: Action LADA 12

Type 1 diabetes can occur at any age and is of variable severity. In adults presenting with the disease, unlike children, it may masquerade as type 2 diabetes, since insulin therapy is not always required [1]. Established autoimmune markers of type 1 diabetes, notably GAD autoantibodies (GADA), have been used to screen individuals presenting with adult-onset diabetes to identify those with type 1 diabetes, including the so-called latent autoimmune diabetes in adults (LADA) [2, 3]. Identifying individuals with adult-onset type 1 diabetes is important as, compared with people with type 2 diabetes, they have a different clinical course and different comorbidities [1]. In adult-onset diabetes, the detection of GADA together with insulinoma-associated antigen-2 autoantibodies (IA-2A) or zinc transporter-8 autoantibodies (ZnT8A) is highly predictive of the need for insulin therapy [4]. Nevertheless, the majority of antibody-positive individuals with adult-onset diabetes present with GADA alone, which may represent either false-positive antibody assay signals in an adult population with a relatively low prevalence of autoimmune type 1 diabetes, or true-positive autoantibodies with high risk for progression to insulin therapy. Antibody characteristics might facilitate diagnosis and estimation of risk for disease progression because high-affinity GADA responses against the middle (amino acids [aa] 235–444) and C-terminal (aa 445–585) domains of the 65 kDa isoform of GAD (GAD65) were associated with type 1 diabetes development in children and adolescents [5]; and high antibody titre [6] and binding to C-terminal (aa 437-585) GAD65-depending epitopes were reported as characteristic features of LADA individuals [7]. In contrast, low-affinity GADA and/or antibody responses restricted to the N-terminal (aa 1–95) domain of GAD65 were not associated with classic type 1 diabetes [5, 8]. Furthermore, using N-terminally truncated (aa 96–585) GAD65 antigen in GADA radiobinding assays improved the specificity for type 1 diabetes [9]. The aim of this study was to evaluate N-terminally truncated (aa 96–585) GAD65 autoantibodies (t-GADA), compared with full-length GAD65 autoantibodies (f-GADA), in a large cohort of adult-onset diabetes individuals with variable clinical phenotype.

Of 1114 participants with adult-onset diabetes, 478 were f-GADA-positive using the NIDDK harmonised radiobinding assay. Of these 478 f-GADA-positive individuals, 55 (11.5%) were t-GADA-negative, thus demonstrating antibody binding restricted to N-terminal GAD65 epitopes (Fig. 1a). Such restricted responses were seen in four (3.2%) of the 126 f-GADA-positive individuals who also had IA-2A and/or ZnT8A (electronic supplementary material [ESM] Fig. 1a), in contrast to 51 (14.5%) of the 352 individuals who only had f-GADA positivity (ESM Fig. 1b; p = 0.0003). Overall, 431 individuals were t-GADA-positive. Antibody titres were strongly correlated between f-GADA and t-GADA assays (r = 0.86, p < 0.0001).

For clinical phenotype, f-GADA-positive individuals were diagnosed with diabetes at a median age of 47.0 years (IQR 38.4–55.7 years), median BMI of 25.6 kg/m² (IQR 22.8–29.4 kg/m²) and 51.2% required insulin therapy (Table 1). For the t-GADA-positive individuals, median age at diagnosis was 46.6 years (IQR 38.0–55.5 years), median BMI 24.9 kg/m² (IQR 22.6–28.8 kg/m²) and 55.3% were on insulin therapy. For the whole cohort, risk of insulin treatment was augmented in t-GADA-positive individuals (OR 4.69 [95% CI 3.57, 6.17]; p < 0.0001 vs t-GADA-negative) compared with f-GADA-positive individuals (OR 3.86 [95% CI 2.95, 5.06]; p < 0.0001 vs f-GADA-negative). This difference was evident irrespective of the disease duration: both for the cohorts assessed at 1 month post-diagnosis (OR 5.20 [95% CI 3.89, 6.94] and 4.42 [3.32, 5.88], respectively) and at 6 months post-diagnosis (OR 4.92 [95% CI 3.61, 6.69] and 4.25 [95% CI 3.13, 5.77], respectively).

Strikingly, when comparing the t-GADA-positive individuals with individuals who were N-terminal GAD65-restricted in antibody binding, i.e. f-GADA-positive but t-GADA-negative, the latter required insulin treatment less often (13.7%; p < 0.0001; Fig. 1b), were older at diagnosis (median age 52.0 years [IQR 41.8–59.8 years]; p = 0.005; Fig. 1c) and were more obese (median BMI 29.9 kg/m² [IQR 26.7–35.9 kg/m²]; p < 0.0001; Fig. 1d). Moreover, individuals with N-terminal GAD65-restricted antibodies were not phenotypically different from individuals who were negative for both f-GADA and t-GADA, i.e. type 2 diabetes individuals (Fig. 1, Table 1).

Finally, when comparing the individuals who were positive (n = 423) or negative (n = 628) in both assays (f-GADA and t-GADA), these positive individuals were younger at diagnosis (p = 0.003), leaner (p < 0.0001) and were more often on insulin treatment (p < 0.0001) (Table 1).

In this study, we found that overall, f-GADA-positive participants and t-GADA-positive participants with adult-onset diabetes had a similar clinical phenotype that distinguished them from GADA-negative participants with type 2 diabetes. Importantly, however, a subgroup of individuals who were positive for f-GADA but negative for t-GADA (i.e. individuals with N-terminal GAD65-restricted GADA) had a similar clinical phenotype to GADA-negative, type 2 diabetic individuals, albeit in relatively low numbers. It is therefore possible that the N-terminal GAD65-restricted GADA, as proposed, does not detect individuals with autoimmune type 1 diabetes, at least using clinical phenotype as a proxy, whilst screening for t-GADA could be helpful for clinical assessment and prediction of insulin therapy.

Screening adult-onset diabetes individuals for GADA to identify autoimmune type 1 diabetes is limited by false-positive and false-negative assay results [1]. Using the t-GADA assay, when compared with the f-GADA assay, we identified a reduced number of GADA-positive individuals (88%) and confirmed a high number (99%) of GADA-negative individuals. Further, we found that the risk of being on insulin treatment within 5 years from diagnosis when positive for t-GADA was higher (OR 4.69) than in those individuals positive for f-GADA (OR 3.86). Those positive for t-GADA, compared with those with N-terminal GAD65-restricted GADA, were enriched for phenotypic features characteristic of individuals with adult-onset type 1 diabetes, including being younger, leaner and with an increased need for insulin treatment.

Differences in GADA epitope binding between LADA individuals with clinical characteristics similar to either type 1 or type 2 diabetes have previously been reported [7]. Individuals with antibodies against epitopes within the N-terminal/middle (aa 1–360) GAD65 region and negative for antibodies against the C-terminal (aa 437–585) GAD65 region showed clinical features that were indistinguishable from those of GADA-negative individuals, whereas the presence of autoantibodies directed to both GAD65 regions identified a subgroup of individuals with low BMI, low basal C-peptide values and the need for insulin treatment [7]. In contrast to this former study and based on our previous data [5, 9], we used the N-terminally truncated (aa 96–585) GAD65 antigen for our t-GADA assay, containing epitopes within the middle and C-terminal GAD65 regions. We believe that the false-positive rate in screening tests can likely be decreased by using this t-GADA assay to identify those at risk of progression to insulin treatment.

In conclusion, our findings suggest that in individuals with adult-onset diabetes, screening for t-GADA augments the clinical phenotype of autoimmune type 1 diabetes. This may have important practical implications given the relatively large number of adult patients who are screened for GADA, both for diabetes classification and prediction of risk for insulin therapy.