Modulation of Functional Activity of INS-VNTR Class I Haplotype Derived from Patients With Type 1 Diabetes

INS-VNTR class I haplotype controls INS expression in thymus that might contribute to insulin tolerance induction.

We investigate the functional activities of differential tandem repeat units (TRUs) derived from insulin autoantibody-positive and -negative patients with type 1 diabetes (T1D).

INS-VNTR class I haplotypes derived from DNA samples from 28 gender-, age-, and human leukocyte antigen (HLA)-matched patients with T1D were polymerase chain reaction amplified and subcloned into pGL3-luciferase vector. The functional activities of the INS-VNTR locus were sequenced for composition and number of TRUs, measured INS-VNTR promoter activities, and AIRE binding capacity. Small molecules that enhance INS-VNTR-luc2 activity were identified.

The mean TRUs (average from both alleles) were significantly longer in IAA- than IAA+ patients (39.8 vs 35.2). INS-VNTR promoter activity responds to the longer TRUs better than the shorter TRUs with AIRE, suggesting that longer TRUs exhibits stronger INS expression capacity potentially favorable for tolerance induction. The binding capacity of AIRE to confer INS transcription from various INS-VNTR haplotypes was determined via ChIP and pull-down assays. The INS-VNTR probe demonstrated differential binding affinity for the AIRE protein corresponding to TRUs, suggesting a differential INS expression in thymus. AIRE protein that binds to INS-VNTR could induce endogenous INS transcript in vitro. All-trans retinoic acid (vitamin A) is capable of stimulating INS-VNTR promoter activity, which provides a valuable option of early treatment of patients with prediabetes with daily vitamin A supplement.

Longer TRUs of the class I INS-VNTR haplotype favors INS expression in thymus and tolerance induction. Vitamin A supplementation could be beneficial for prevention of insulin autoimmunity at the early stages of T1D onset.