Maturation of the humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 diabetes.

Abstract

GAD is a major target of autoimmunity in preclinical t y p e 1 diabetes. Here we examine the maturation of the humoral response to GAD epitopes sequentially from birth to diabetes onset or current follow-up in 29 GAD antibody (GADA) + o ffspring of parents with diabetes from the BABYDIAB Study. Antibodies were measured against GAD65, GAD67, and GAD65/67 chimeras by radiobinding assay. In 28 of 29 offspring, the first GADAs contained reactivity against epitopes within GAD65 residues 96-444, suggesting that the middle GAD65 region is a primary target of GAD humoral autoimmunity. In 7 of these 28 offspring, initial antibody reactivity was against all epitope regions tested (middle GAD65, COOH-terminal GAD65 residues 445-585, NH 2 -t e r m i n a l GAD65 residues 1-95, and GAD67); in 16 offspring, reactivity was to middle and COOH-terminal GAD65 epitopes, and in 5 offspring, reactivity was only to the middle GAD65 epitopes. The single offspring without middle GAD65 reactivity had antibodies to the NH 2 -terminal epitopes in the absence of all other islet autoimmunity. Subsequent GADA epitope spreading was frequent and seen in 10 of 15 offspring with informative follow-up samples. Spreading was mostly (eight cases) to NH 2 -t e r m i n a l GAD65 epitopes. In two offspring, spreading to new epitopes was found when antibody titers to GAD65 and early epitopes were declining, suggesting determinants p e c i fic regulation of the humoral response. None of the GADA reactivities nor any changes in reactivity over time were specifically associated with diabetes onset. The findings suggest that the humoral autoimmune response to GAD found in childhood is dynamic, is initially against epitopes within the middle portion of GAD65, and spreads to epitopes in other regions of GAD65 and GAD67. D i a b e t e s 4 9 :2 0 2-208, 2000

GAD autoantibodies and epitope reactivities persist after diagnosis in latent autoimmune diabetes in adults but do not predict disease progression: UKPDS 77

“…GAD65 epitope recognition Recognition of conformational epitopes of GAD65 has been proposed as a marker for maturation of the humoral response in preclinical phases of type 1 diabetes [10,31] and suggested as a method for differentiation of disease and/or progression in adult-onset diabetes [12,[31][32][33]. In our cohort, the C-terminal and middle regions of GAD65 contained the most frequently targeted epitopes (in more than 50% of patients) for samples taken at all time points after diagnosis, whereas N-terminal reactivity was uncommon (<9%).…”

“…In this group of 242 patients, comparisons were made of GADA levels determined previously in samples taken at diagnosis with those taken at 0.5 years in this study and at 3 and 6 years post-diagnosis (see ESM Fig. 2) Plasma GADA levels were determined with a radiobinding assay using 35 S-labelled human GAD65 as antigen which was validated in the 2003 Diabetes Antibody Standardization Program (DASP 2003) [10,11]. GAD65 was produced by in vitro transcription/translation of human GAD65 (also known as GAD2) cDNA using a rabbit reticulocyte lysate (Promega, Madison, WI, USA) and L-[…”

“…The fusion proteins comprised GAD65 epitopes: (1) at the N-terminal (GAD65 1-95 /GAD67 102-593 ); (2) in the middle region (GAD67 1-243 /GAD65 235-442 /GAD67 443-593 ); or (3) at the C-terminal (GAD67 1-452 /GAD65 445-585 ) [10,12]. Reactivities to GAD65 epitopes and GAD67 were expressed as arbitrary units relative to a standard prepared from samples from patients with type 1 diabetes or Stiff-Person syndrome.…”

GAD autoantibody affinity in schoolchildren from the general population

“…GADA epitope measurements GADA epitope specificity was determined using radiobinding assays on [ 35 S]-methioninelabelled GAD65/67 kDa isoform of GAD (GAD67) chimeric proteins as previously described [17]. Thresholds for positivity were defined as the upper limit in the sera of 50 control participants.…”

“…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].…”