Humoral autoreactivity to an alternatively spliced variant of ICA512/1A-2 in Type I diabetes

Aims/hypothesis. The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 M-r type 1 transmembrane glyc oprotein in beta cells and is a major autoantigen of Type I (insulin-depend ent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule. Methods. Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 1 3 which produces a secreted form lacking aa 557-629 including the transmemb rane domain (aa 577 to 600). The alternatively spliced protein is detectabl e by western blotting in normal islets and translated into a protein that i s processed to a series of soluble forms of 25,000-35,000 M-r. Radioimmuno- precipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. Results. The assays showed that ICA512.bdc and ICA512(604-979) gave the bes t discrimination between diabetic and control sera. With ICA512(604-979) a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5 % vs 63.2% of prediabetic /new-onset and 25.0 vs 13.9% in patients with diabetes > 20 years). Convers ely, a small proportion (3 % recent-onset and 6% > 20 years) had antibodies to ICA512.bdc but not ICA512(604-979). Conclusion/interpretation. Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the Delta exon 13 variant is probably secreted by the beta cell, it could be recognized b y the cellular and humoral arm of the immune system in the absence of cellu lar damage.