Conformation-dependent GAD65 autoantibodies in diabetes

Conformation-dependent autoantibodies directed against GAD65 are markers of Type 1 diabetes. In this study we aimed to determine whether the substitution of GAD65 with GAD67 amino acids would affect the binding of conformation-dependent GAD65 autoantibodies.

We used PCR-based site-directed mutagenesis to generate a series of mutated GAD65 cDNA constructs in which specific GAD65 coding sequences for regions of the protein critical for autoantibody binding were replaced with GAD67 coding sequences.

The introduction of a point mutation at position 517, substituting glutamic acid with proline, markedly reduced the binding of disease-associated GAD65 antibodies. The binding of GAD65 antibodies to the E517P mutant was reduced in the sera of all newly diagnosed Type 1 diabetes patients ( n=85) by a mean of 72% ( p<0.0001) compared with binding to wild-type GAD65. Patients with latent autoimmune diabetes in adults ( n=24) showed a similar reduction in binding (79% reduction, p<0.0001). First-degree relatives who subsequently progressed to Type 1 diabetes ( n=12) showed a reduction in binding of 80% compared with a reduction of only 65% among relatives who had not progressed to disease ( n=38; p=0.025). In healthy GAD65Ab-positive individuals who did not progress to diabetes during a 9-year follow-up period ( n=51), binding to GAD65-E517P was reduced by only 28% compared with binding to wild-type GAD65.

Differences in autoantibody binding to wild-type GAD65 versus GAD65-E517P may provide predictive information about Type 1 diabetes risk beyond that provided by the presence or absence of GAD65 autoantibodies. Lack of binding to mutant GAD65-E517P defines GAD65-positive individuals who are at higher risk of developing diabetes.