GAD65 antibody isotypes and epitope recognition during the prediabetic process in siblings of children with type I diabetes

Abstract

SUMMARYWe observed 42 initially non-diabetic siblings of affected children to characterize the humoral immune response to the 65 kDa isoform of glutamic acid decarboxylase (GAD65) in preclinical type I diabetes. During the observation period with a mean duration of 9·6 years 21 siblings progressed to type I diabetes. The humoral immune response to GAD65 was observed initially as a simultaneous response to the middle (M) and carboxy (C)-terminal regions of the GAD65 molecule in most cases, and if the response was restricted initially to the middle region, it spread rapidly to the C-terminal domain and in a few cases later to the amino (N)-terminal domain. There was some heterogeneity in the GAD65 isotype response, but it was composed mainly of antibodies of immunoglobulin (Ig) G1 subclass. Responses of IgG2-, IgG4-, IgM-and IgA-GAD65Ab were observed frequently, whereas IgE-and IgG3-GAD65Ab responses were seen more rarely. Initially, the non-progressors tended more often to have IgG2-and IgG4-GAD65Ab than the progressors. As a sign of a dynamic process a significant isotype spreading was seen for IgG2-GAD65Ab ( P < 0·05) and close to significant for IgM ( P = 0·06) among progressors and for IgM-GAD65Ab ( P < 0·05) among non-progressors during the observation period. This study failed to identify any GAD65 epitope-or isotype-specific antibody reactivity that could be used as a marker for progression to disease, as such progression was not associated with any specific changes in reactivity over time. Our findings indicate that epitope-and isotype-specific GAD65 antibodies are hardly capable of separating progressors from non-progressors among GAD65Ab-positive first-degree relatives of children with type I diabetes.

Autoantibodies in Diabetes

“…This series of events is poorly documented in spontaneously diabetic laboratory rodents, such as the NOD mouse or the BB rat, because neither animal has an autoantibody response of the character and magnitude that is observed in human type 1 diabetes. In humans, limited information about GAD65Ab isotypes at onset and in the prodromal period is available (7,8). The common assay used for the detection of autoantibodies is based on immunoprecipitation using Protein A Sepharose.…”

“…Both multiple epitopes (7,8,70,75,78) and the reaction to the juxtamembrane region have been shown to be linked with progression to diabetes (7,78). In a longitudinal study of first-degree relatives, intramolecular epitope spreading was only observed among the progressors, while some of the nonprogressors showed a decrease in the number of epitopes bound (75).…”

“…One study (7) in a few first-degree relatives of type 1 diabetic patients failed to identify any GAD65 epitope-or isotype-specific antibody reactivity that could be used as a marker for progression to disease. This is in contrast to another study (8), which showed that epitope-and isotypespecific autoantibodies were capable of separating infant (children born to type 1 diabetic mothers) progressors from nonprogressors.…”

Natural History of Type 1 Diabetes

“…With respect to GADAs, epitope spreading is frequent (17). Autoantibody reactivity is initially directed against epitopes within the middle region (residues 235-444) and the COOHterminal region (residues 440 -585) of GAD65, indicating that either a rapid spreading of reactivity or simultaneous immunization against distinct GAD65 regions can occur (17,18). Reactivity to NH 2 -terminal GAD65 epitopes in children is less common, weaker, and usually appears after that against central and COOH-terminal epitopes.…”

GAD autoantibody affinity in schoolchildren from the general population

“…GADA often appear in older age groups and are preferentially associated with HLA-DRB1*03 haplotypes [11]. The GADA level alone does not predict progression to diabetes, and controversial reports exist with respect to the relevance of GADA epitope reactivity to diabetes progression [12][13][14][15][16][17].…”