Characteristics of in-vitro phenotypes of glutamic acid decarboxylase 65 autoantibodies in high-titre individuals

Abstract

Previous studies have indicated phenotypical differences in glutamic acid decarboxylase 65 autoantibodies (GADA) found in type 1 diabetes (T1D) patients, individuals at risk of developing T1D and stiff-person syndrome (SPS) patients. In a Phase II trial using aluminium-formulated GAD(65) (GAD-alum) as an immunomodulator in T1D, several patients responded with high GADA titres after treatment, raising concerns as to whether GAD-alum could induce GADA with SPS-associated phenotypes. This study aimed to analyse GADA levels, immunoglobulin (Ig)G1-4 subclass frequencies, b78- and b96·11-defined epitope distribution and GAD(65) enzyme activity in sera from four cohorts with very high GADA titres: T1D patients (n = 7), GAD-alum-treated T1D patients (n = 9), T1D high-risk individuals (n = 6) and SPS patients (n = 12). SPS patients showed significantly higher GADA levels and inhibited the in-vitro GAD(65) enzyme activity more strongly compared to the other groups. A higher binding frequency to the b78-defined epitope was found in the SPS group compared to T1D and GAD-alum individuals, whereas no differences were detected for the b96·11-defined epitope. GADA IgG1-4 subclass levels did not differ between the groups, but SPS patients had higher IgG2 and lower IgG4 distribution more frequently. In conclusion, the in-vitro GADA phenotypes from SPS patients differed from the T1D- and high-risk groups, and GAD-alum treatment did not induce SPS-associated phenotypes. However, occasional overlap between the groups exists, and caution is indicated when drawing conclusions to health or disease status.

Figure 1

Serum glutamic acid decarboxylase antibody (GADA) titres (U/ml) in stiff-person syndrome (SPS) (circles, n = 12), type 1 diabetes mellitus (T1D) (squares, n = 7), glutamic acid decarboxylase (GAD)-alum (rhombuses, n = 9) and high-risk (triangles, n = 6) groups. Empty circles in the SPS group (n = 8) represent individuals with co-existent T1D, whereas empty triangles in the high-risk group (n = 3) represent individuals who developed T1D after sampling. Significant differences are indicated as P-values and horizontal lines represent the median.

Figure 2

Recombinant human glutamic acid decarboxylase (GAD)₆₅ in-vitro enzyme activity in the presence of sera from stiff-person syndrome (SPS) (circles, n = 12), type 1 diabetes mellitus (T1D) (squares, n = 7), GAD-alum (rhombuses, n = 9) and high-risk (triangles, n = 6) groups. Open circles in the SPS group (n = 8) represent individuals with co-existent T1D, whereas open triangles in the high-risk group (n = 3) represent individuals who developed T1D after sampling. Results are expressed as a percentage of maximum GAD₆₅ enzyme activity. Significant differences are indicated as P-values and horizontal lines represent the median.

Figure 3

Correlation between glutamic acid decarboxylase antibody (GADA) titres and glutamic acid decarboxylase (GAD)₆₅ enzyme activity in stiff-person syndrome (SPS) (a) and type 1 diabetes mellitus (T1D) patients (b), GAD-alum treated T1D patients (c) and high-risk T1D individuals (d). Open circles in the SPS group (n = 8) represent individuals with co-existent T1D, whereas open triangles in the high-risk group (n = 3) represent individuals who developed T1D after sampling. Significant differences or trends are indicated as P-values, and the correlation coefficient as r.

Figure 4

(a,b) Binding to GAD₆₅ in the presence of rFab b78 (a) and rFab b96·11 (b) presented as a ratio of competed/non-competed in stiff-person syndrome (SPS) (circles, n = 12), type 1 diabetes mellitus (T1D) (squares, n = 7), glutamic acid decarboxylase (GAD)-alum (rhombuses, n = 9) and high-risk (triangles, n = 6) groups. Open circles in the SPS group (n = 8) represent individuals with co-existent T1D, whereas open triangles in the high-risk group (n = 3) represent individuals who developed T1D after sampling. A higher binding to GAD₆₅ in the presence of rFab indicates a lower proportion of glutamic acid decarboxylase antibody (GADA) binding to the respective epitope. Samples with a calculated value below the 85% cut-off limit, represented as a dotted line, were regarded as positive for binding to the respective epitope. Significant differences are indicated as P-values and horizontal lines represent the median.

Figure 5

Serum glutamic acid decarboxylase antibody (GADA) IgG 1–4 subclass distribution in stiff-person syndrome (SPS) (n = 10), type 1 diabetes mellitus (T1D) (n = 7), glutamic acid decarboxylase (GAD)-alum (n = 9) and high-risk (n = 6) groups. Results are expressed as the relative contribution of each subclass (% of total GADA) and positivity of each sample was calculated by subtraction of the mean cpm value plus three times the standard deviation (s.d.) obtained for the negative control. Horizontal lines represent the median.