Autoantibodies Directed Toward a Novel IA-2 Variant Protein Enhance Prediction of Type 1 Diabetes

Abstract

We identified autoantibodies (AAb) reacting with a variant IA-2 molecule (IA-2var) that has three amino acid substitutions (Cys²⁷, Gly⁶⁰⁸, and Pro⁶⁷¹) within the full-length molecule. We examined IA-2var AAb in first-degree relatives of type 1 diabetes (T1D) probands from the TrialNet Pathway to Prevention Study. The presence of IA-2var-specific AAb in relatives was associated with accelerated progression to T1D in those positive for AAb to GAD65 and/or insulin but negative in the standard test for IA-2 AAb. Furthermore, relatives with single islet AAb (by traditional assays) and carrying both IA-2var AAb and the high-risk HLA-DRB1*04-DQB1*03:02 haplotype progress rapidly to onset of T1D. Molecular modeling of IA-2var predicts that the genomic variation that alters the three amino acids induces changes in the three-dimensional structure of the molecule, which may lead to epitope unmasking in the IA-2 extracellular domain. Our observations suggest that the presence of AAb to IA-2var would identify high-risk subjects who would benefit from participation in prevention trials who have one islet antibody by traditional testing and otherwise would be misclassified as "low risk" relatives.

Figure 1

A: Schematic representation of IA-2var (GeneBank accession number: MN071399). The vertical lines represent the amino acid substitutions: SNP₁ (S→C), SNP₂ (D→G), and SNP₃ (S→P). B: Sequence data derived from the IA-2var construct. A total of four SNPs were identified, three of which (shown) are nonsynonymous SNPs. C: Predicted three-dimensional structural models of native IA-2 (gold) and IA-2var (blue). C1: Predicted structures are overlay and matched with UCSF Chimera (version 1.13.1). C2: Zoom for region showing residues LLSSRPG for native IA-2 (gold) and SNP₁ residues LLSCRPG for IA-2var (blue). C3: Zoom for region showing residues RQQDKER for native IA-2 (gold) and SNP₂ residues RQQGKER for IA-2var (blue). C4: Zoom for region showing residues SPSSHSS for native IA-2 (gold) and SNP₃ residues SPSPHSS for IA-2var (blue). Bolded amino acid one-letter abbreviations correspond to amino acid substitutions.

Figure 2

IA-2var AAb are associated with high risk of T1D progression in relatives with single or multiple islet AAb at screening. A: Progression to T1D in relatives who were negative for ICA512bdc AAb and were selected for having a single AAb (GAD65 AAb or IAA) in the absence (dashed line) or presence (solid line) of IA-2var AAb (P < 0.0001). B: Progression to T1D in relatives who were negative for ICA512bdc AAb and were selected for having multiple AAb (GAD65 AAb and IAA) in the absence (dashed line) or presence (solid line) of IA-2var AAb (P = 0.001). CR, cumulative risk; NEG, negative; POS, positive.

Figure 3

IA-2var AAb improve prediction of T1D in relatives testing negative for the conventional IA-2 AAb assay (ICA512bdc AAb). A: Relatives selected for being negative for ICA512bdc AAb in the absence (dashed line) or presence (solid line) of IA-2var AAb (P < 0.0001). B: Relatives who were negative for IA-2var AAb in the absence (dashed line) or presence (solid line) of AAb against ICA512bdc (P = 0.3). C and D: Progression to T1D in relatives in relation to the absence (dashed line) or presence (solid line) of IA-2var AAb (C) and progression to T1D in relatives in the absence (dashed line) or presence (solid line) of ICA512bdc AAb (D). CR, cumulative risk; NEG, negative; POS, positive.

Figure 4

IA-2var AAb can be detected in relatives who are negative for traditional islet AAb at screening (ICA512bdc, GAD65, and mIAA). A: 7.8% (44 of 566) of the relatives who developed T1D (progressors) were negative for ICA512bdc but negative for IA-2var AAb. B: 2.2% (25 out of 1,120) of ICA512bdc AAb–negative nonprogressors were IA-2var AAb positive. C and D: 14.2% (81 of 566) of progressors were IA2var AAb positive and GAD65 AAb negative (C), and 29.7% (168 of 566) of the progressors were IA-2var AAb positive but mIAA negative (D). E: In seronegative relatives at screening (negative for ICA512bdc, GAD65 AAb, IAA), the presence of IA-2var AAb (solid line) still conferred a higher risk of progression to T1D compared with those negative for IA-2var AAb (dashed line) (P = 0.001). CR, cumulative risk; NEG, negative; POS, positive.

Figure 5

IA-2var AAb and HLA DQ and DR genotypes confer a significant T1D risk in relatives with single and multiple islet AAb tested by traditional antibody assays. All of these subjects lacked ICA512bdc AAb. A: Relatives with single islet AAb (GAD65 AAb or IAA) carrying both IA-2var AAb and the HLA-DRB1*04-DQB1*0302 haplotype (P < 0.0001) (solid line). Relatives with single islet AAb (GAD65 AAb or IAA) carrying the HLA-DRB1*04-DQB1*0302 haplotype who were negative for IA-2var AAb (dotted line). Relatives with single islet AAb (GAD65 AAb or IAA) who were negative for IA-2var AAb and lacked the HLA-DRB1*04-DQB1*0302 haplotype (dashed line). B: Relatives with multiple islet AAb (GAD65 AAb and IAA) carrying both IA-2var AAb and the HLA-DRB1*04-DQB1*0302 haplotype (P = 0.02) (solid line). Relatives with multiple islet AAb (GAD65 AAb and IAA) carrying the HLA-DRB1*04-DQB1*0302 haplotype who were negative for IA-2var AAb (dotted line). Relatives with multiple islet AAb (GAD65 AAb and IAA) who were negative for IA-2var and lacked the HLA-DRB1*04-DQB1*0302 haplotype (dashed line). CR, cumulative risk; NEG, negative; POS, positive.

Figure 6

The presence of IA-2var AAb conferred a high risk of T1D progression in both relatives younger and older than 14 years of age who were positive at screening for single or multiple islet AAb by traditional antibody testing. All of these subjects lacked ICA512bdc AAb. A: Progression to T1D in relatives younger than 14 years of age who were positive for single islet AAb at screening (GAD65 AAb or IAA) in the absence (dashed line) or presence (solid line) of IA-2var AAb (P < 0.0001). B: Progression to T1D in relatives younger than 14 years of age who were positive for multiple islet AAb at screening in the absence (dashed line) or presence (solid line) of IA-2var AAb (P = 0.04). C: Progression to T1D in relatives older than 14 years of age who were positive for single islet AAb at screening (GAD65 AAb or IAA) in the absence (dashed line) or presence (solid line) of IA-2var AAb (P < 0.0001). D: Progression to T1D in relatives older than 14 years of age who were positive for multiple islet AAb at screening in the absence (dashed line) or presence (solid line) of IA-2var AAb (P = 0.03). CR, cumulative risk; NEG, negative; POS, positive.