The present study was initiated to resolve a paradoxical finding that two constructs bearing the COOH-terminal antigenic region of ZnT8 with or without the NH2
-terminus (supplementary Fig. 1) were recognized in a differential fashion by subsets of type 1 diabetes new-onset sera (3
). The constructs were derived from different cDNAs and subsequently shown to encode the Arg (C-probe) or Trp (NC-probe) variants of aa325
. To further explore this phenomenon, assays were performed in sera from newly diagnosed type 1 diabetic patients using COOH-terminal ZnT8 probes bearing Arg, Trp, or Gln at aa325
: 259 of 421 individuals (61.5%) reacted to at least one probe, with the highest response recorded in reaction to the Arg variant (53.2%) followed by Trp (43.9%) and Gln (33.7%) (P
< 0.0001, χ2
). Analysis of the overlap in responses () shows that some individuals react to the Arg or Trp probes alone and very rarely to Gln alone; 29.7% of individuals reacted to all three probes.
A comparison of the levels of autoantibody reactivity to the Arg and Gln probes () showed that the majority of individuals either reacted equivalently to the probes (falling within the bounds of the diagonal of the x-y plot ± 3 SD) or responded to the Arg probe alone. Trp and Gln reactivities () were similarly separated. Of the 34 patients who reacted equivalently to Arg and Trp probes (within the bounds of the diagonal ± 3 SD of ), 29 (85.3%) had an equivalent response as determined by the Gln probe. This indicated that for these individuals, the aa at position 325 was not a determinant of autoantibody reactivity. A series of preabsorption experiments was therefore performed using peptides and recombinant proteins as competing ligands (). Selected type 1 diabetes sera that reacted with the Arg probe alone were blocked by recombinant NUS-C-term Arg325 protein but not by NUS-C-term Trp325 or NUS-C-term Gln325. Similarly, Trp-only responses were blocked by NUS-C-term Trp325 but not NUS-C-term Arg325 or NUS-C-term Gln325. Sera that reacted equivalently to Arg, Trp, and Gln probes were blocked by any of the NUS-C-term ZnT8 recombinants. Overlapping 20-mer peptides spanning the ZnT8 COOH-terminal from 268–369 did not compete for reactivity, suggesting that the epitopes were conformational rather than linear. Overall, these results suggest that ZnT8A reactivity could be accounted for by three classes of conformational epitopes: one for which Arg325 was an essential determinant, a second Trp325 restricted, and a third not affected by aa325.
FIG. 2. Preabsorption of autoantibodies with recombinant proteins. Single sera samples were selected from hCArg-restricted sera, hCTrp-restricted sera, or samples that react equivalently with hCGln, hCArg, and hCTrp probes. Samples were preincubated without (-) (more ...)
The relationship between ZnT8 autoantibody reactivity and genetic variation at the SLC30A8
locus was examined using the SNP (rs13266634) encoding the Arg/Trp325
variant and two adjacent noncoding SNPs identified in a type 2 diabetes genome-wide association study (6
), rs2466295, located 259 bp distally in the 3′ UTR, and rs6469675, located 19635 bp proximally in intron 2. The minor allele frequency (MAF) for rs13266634 in our type 1 diabetic population of 0.266 (n
= 351) approximated the reference frequency of 0.256 (n
= 168) for Europeans in the NLM SNP database (http://www.ncbi.nlm.nih.gov/SNP/snpref.cgi?rs=13266634
). The distribution of genotypes (55.3% CC, 36.2% CT, and 8.5% TT) was consistent with Hardy-Weinberg distribution (53.9, 39.0, and 7.1, respectively). Similar correlations were observed for the MAF for rs6469675 (0.285 vs. 0.220 in our study vs. the NLM SNP database, respectively) and rs2466295 (0.361 vs. 0.407).
The specificity of the ZnT8A response reflected the rs13266634 genotype (), with little or no association observed with the adjacent rs2466295 and rs6469675 SNPs. The ZnT8A response assessed by the Gln probe showed no significant variation with the rs13266634 genotype, whereas responses to the Arg probe were highest in CC homozygotes, lowest in TT homozygotes, and intermediate in the heterozygote group. Conversely, responses to the Trp probe were highest in TT homozygotes, lowest in CC, homozygotes, and intermediate in heterozygotes. An even stronger relationship with genotype was seen in the groups having only Arg325- and Trp325-restricted responses. Arg325-only responses were observed only in individuals bearing the rs13266634 C-allele, with a 4.2-fold higher frequency in homozygotes than heterozygotes. With one exception, all Trp325-restricted responses were associated with the rs13266634 T-allele, with a 10.2-fold higher frequency in homozygotes than heterozygotes. The single Gln325-restricted ZnT8A patient () was genotyped CC, confirmed by sequencing. The small number of sera positive for both Trp325 and Arg325 but not Gln325 probes were associated with heterozygote genotypes (11 of 13 cases) as expected (data not shown). The prevalence of insulin autoantibody, GAD antibody, or IA-2 antigen response did not change as a function of rs13266634 genotype.
Prevalence of autoantibody in relation to rs13266634 genotype
The median age of onset of disease in the genotyped individuals was 11.2 years (range 0.6–58), with more than half (57.3%) diagnosed between ages 8 and 16 years and 88.9% before age 18 years. A frequency distribution analysis based on binning at 4-year intervals showed no statistically significant difference in the representation of the CC, CT, and TT genotypes at any age of onset, though a trend was observed for a higher CC and lower CT genotype frequency in the youngest onset group (0.6–4 years) compared with that of a 5- to 15-year-old reference group (Fishers exact test, P = 0.24; n = 219) ().
FIG. 3. Prevalence of genotypes, autoantibodies to ZnT8, and other antigens in relation to age ofclinical diagnosis. Data from 335 individuals were subdivided in groups 4 years apart and analyzed with respect to distribution of genotypes (A); presence of insulin, (more ...)
The prevalence of ZnT8A measured with Gln325, Arg325, and Trp325 COOH-terminal probes increased with increasing age of onset, reached a plateau at 8–16 years, and then fell (). Arg325- and Trp325-restricted responses were observed in all age-groups, but the low numbers of positive individuals did not permit ascertainment of changes in prevalence and levels of reactivity in association with age (). The autoantibody responses to insulin, GAD65, and IA-2 showed characteristic changes in prevalence relative to age of onset of disease, insulin autoantibody prevalence being highest in younger onset patients, IA-2 antibody tending to be higher in adolescents than children, and GAD antibody showing little variation ().