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

Abstract

Context

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

Objective

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

Methods

INS-VNTR class I haplotypes derived from 28 gender, age, and HLA matched T1D patients’ DNA samples were PCR amplified and subcloned into pGL3-luciferase vector. The functional activities of INS-VNTR locus were sequenced for composition and number of TRUs, measured INS-VNTR promoter activities, and AIRE binding capacity. Identify small molecules that enhance INS-VNTR-luc2 activity.

Results

The mean TRUs (average from both alleles) were significantly longer in IAA^- than IAA⁺ patients (39.8 versus 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 haplotype was determined via ChIP and pull-down assays. INS-VNTR probe demonstrated differential binding affinity for AIRE protein corresponding to TRUs suggesting a differential INS expression in thymus. AIRE protein binds to INS-VNTR could induce endogenous INS transcript in vitro. ATRA (vitamin A) is capable of stimulating INS-VNTR promoter activity, which provides a valuable option of early treatment of pre-diabetic patients with daily vitamin A supplement.

Conclusion

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

Insulin autoimmunity, VNTR, AIRE, tolerance, T1D, ATRA

Accepted manuscripts

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