Genetic association of zinc transporter 8 (ZnT8) autoantibodies in type 1 diabetes cases
- First Online:
- Cite this article as:
- Howson, J.M.M., Krause, S., Stevens, H. et al. Diabetologia (2012) 55: 1978. doi:10.1007/s00125-012-2540-2
- 785 Downloads
Autoantibodies to zinc transporter 8 (ZnT8A) are associated with risk of type 1 diabetes. Apart from the SLC30A8 gene itself, little is known about the genetic basis of ZnT8A. We hypothesise that other loci in addition to SLC30A8 are associated with ZnT8A.
The levels of ZnT8A were measured in 2,239 British type 1 diabetic individuals diagnosed before age 17 years, with a median duration of diabetes of 4 years. Cases were tested at over 775,000 loci genome wide (including 53 type 1 diabetes associated regions) for association with positivity for ZnT8A. ZnT8A were also measured in an independent dataset of 855 family members with type 1 diabetes.
Only FCRL3 on chromosome 1q23.1 and the HLA class I region were associated with positivity for ZnT8A. rs7522061T>C was the most associated single nucleotide polymorphism (SNP) in the FCRL3 region (p = 1.13 × 10−16). The association was confirmed in the family dataset (p ≤ 9.20 × 10−4). rs9258750A>G was the most associated variant in the HLA region (p = 2.06 × 10−9 and p = 0.0014 in family cases). The presence of ZnT8A was not associated with HLA-DRB1, HLA-DQB1, HLA-A, HLA-B or HLA-C (p > 0.05). Unexpectedly, the two loci associated with the presence of ZnT8A did not alter risk of having type 1 diabetes, and the 53 type 1 diabetes risk loci did not influence positivity for ZnT8A, despite them being disease specific.
ZnT8A are not primary pathogenic factors in type 1 diabetes. Nevertheless, ZnT8A testing in combination with other autoantibodies facilitates disease prediction, despite the biomarker not being under the same genetic control as the disease.
KeywordsAutoantibodyFCRL3HLAMHCSLC30A8Type 1 diabetesZinc transporter 8
Genome-wide association study
Autoantibodies to insulinoma-associated antigen-2
Autoantibodies to insulin
Islet cell autoantibodies
Single nucleotide polymorphism
Type 1 diabetes genetics consortium
Wellcome Trust Case Control Consortium
Autoantibodies to zinc transporter 8
Autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA) and islet cell autoantibodies (ICA) have long been established as associated with the development of type 1 diabetes, with over 90% of newly diagnosed cases positive for at least one of these autoantibodies [1–4]. Most recently, autoantibodies to the pancreatic beta cell-specific protein, zinc transporter 8 (ZnT8A), have also been shown to be associated with type 1 diabetes . Over 60% of newly diagnosed cases are positive for ZnT8A, and 4% of cases are positive for ZnT8A only [5–7], thus suggesting their utility as a predictive and diagnostic marker in type 1 diabetes [8–10]. The percentage of newly diagnosed cases positive for ZnT8A (as well as the magnitude of the ZnT8A titre) has been positively correlated with an older age at diagnosis of type 1 diabetes in children [5, 11] and has been shown to decline following diagnosis of type 1 diabetes [12, 13]. The same correlations have also been found with IA-2A and GADA .
The ZnT8 protein is encoded by the SLC30A8 gene, which is convincingly associated with type 2 diabetes risk at the single nucleotide polymorphism (SNP) rs13266634C>T, an amino acid substitution R325W [15–17]. Although this amino acid variant determines the epitope specificity of ZnT8A in type 1 diabetes, either ZnT8RA or ZnT8WA [8, 11], its association with risk of the disease itself is less clear [18–20].
ZnT8RA have been reported as associated with HLA-DQB1*0302 genotypes  and both ZnT8RA and ZnT8WA have been correlated with HLA-DQB1*0604 haplotypes . No other genetic associations have been reported with ZnT8A to date. Given the association of ZnT8A with type 1 diabetes, the 52 non-HLA regions of the genome associated with type 1 diabetes (www.t1dbase.org, accessed 13 December 2011), have a high prior probability of also being associated with ZnT8A positivity. Therefore, we measured ZnT8A (R and W epitopes) in 2,239 British individuals with type 1 diabetes and tested the known type 1 diabetes regions for association with presence of ZnT8A. A genome-wide association study (GWAS) was also performed to further test for genetic determinants of ZnT8A. Finally, we re-examined the association of rs13266634 in SLC30A8 with ZnT8A and type 1 diabetes.
Phenotypes of the type 1 diabetic cases by positivity for ZnT8A status
n, all samples
Age at diagnosis
Duration of diabetes
ZnT8A were measured in serum by protein A radiobinding assays as previously described  using COOH-terminal (aa268-369) constructs of the ZnT8 R325 (ZnT8RA) and W325 (ZnT8WA) variants, respectively. cDNAs for the preparation of radioligands by in vitro transcription–translation were kind gifts of V. Lampasona (San Raffaele Scientific Institute, Milan, Italy). Samples were considered ZnT8A positive if antibodies to at least one of the ZnT8 variants (ZnT8RA and/or ZnT8WA) were found. Performance in the Diabetes Antibody Standardization Program 2009 workshop is shown as ‘laboratory 121’ in a published report . Details of the GADA and IA-2A assays, and the ZnT8A assay in T1DGC families, have been reported previously [14, 21, 22].
The HLA-DRB1, HLA-DQB1, HLA-A, HLA-B and HLA-C genes were typed at four digit resolution using Dynal RELI SSO assays (Invitrogen, Paisley, UK) as detailed elsewhere . A subset of cases was genotyped at HLA-DRB1 and HLA-DQB1 using Roche Molecular Systems SSO reverse dot blot technology (Roche Molecular Systems, Pleasanton, CA, USA). The T1DGC GWAS genotyped 487,592 SNPs that passed quality control checks (2,420 within the extended MHC) using the Illumina 550K Infinium platform  (Illumina, San Diego, CA, USA); and 362,838 SNPs (1,687 within the extended MHC) study were genotyped using the Affymetrix GeneChip Human Mapping 500K Array set (Affymetrix, Santa Clara, CA, USA) for the Wellcome Trust Case Control Consortium (WTCCC) GWAS ; 75,181 were common to both platforms. Follow-up SNPs were genotyped blind to ZnT8A status using the TaqMan 5′ nuclease assay (Applied Biosystems, Warrington, UK) according to the manufacturer's protocols. All genotyping in the family samples used TaqMan. Two operators scored genotypes blind to disease status.
All statistical analyses were performed in R (www.r-project.org, accessed 13 December 2011)  or STATA (www.stata.com, accessed 13 December 2011). Association with ZnT8A (including R and W epitopes) was assessed using logistic regression models, with autoantibody positivity as the outcome variable and genotype as the predictor. Covariates, age at diagnosis and duration of diabetes were included in the regression models to account for the decrease in positivity for ZnT8A with duration of diabetes and the increase in the number of cases positive for ZnT8A with increasing age at diagnosis. For family analyses, robust variance estimates were used to account for non-independence within families . Missing genotypes were imputed in the GWAS datasets using a common set of 1,500 controls genotyped on both platforms as reported previously . Imputed genotypes were weighted by their posterior expectation in the regression models. To assess which autoantibody was associated with rs7522061, ZnT8A and IA-2A were used as predictors of rs7522061 genotype in regression models.
Positivity for ZnT8A in type 1 diabetes cases by quartiles of the duration of diabetes distribution
A subset of 1,307 cases with ZnT8A measurements had previously been tested for GADA and IA-2A [14, 21]. Of these, just 277 (21%) were negative for all three autoantibodies and 28 were only positive for ZnT8A. There was a strong association of positivity for ZnT8A with both IA-2A and GADA positivity, after accounting for age at diagnosis and duration of disease (OR 4.45 [95% CI 3.32, 5.99] p = 8.46 × 10−26 and OR 1.63 [95% CI 1.24, 2.14] p = 5.04 × 10−4, respectively); 81% of ZnT8A positives were also positive for IA-2A, whereas 63% were positive for GADA.
ZnT8A association in type 1 diabetes regions
Association of positivity for ZnT8A with rs7522061 T > C in FCRL3
OR [95% CI]
1.82 [1.57, 2.10]
1.13 × 10−16
1.70 [1.45, 1.98]
8.13 × 10−12
1.65 [1.42, 1.91]
1.74 × 10−11
1.50 [1.22, 1.84]
1.20 × 10−4
The association at FCRL3 was confirmed in up to 855 affected family members of white European ancestry. Confirmation of association with ZnT8A was obtained at rs7522061, rs7528684 and rs11264798 (p = 1.20 × 10−4, 3.14 × 10−4 and 9.20 × 10−4, respectively) with the same direction of effect as in the British cases (OR for the T allele at rs7522061, 1.50 [95% CI 1.22, 1.84]; Table 3).
Association of ZnT8A with SLC30A8
Association of rs13266634 in SLC30A8 with positivity for ZnT8A in 2,191 type 1 diabetes cases
OR [95% CI]
OR [95% CI]
OR [95% CI]
1.07 [0.93, 1.24]
2.38 [2.02, 2.80]
0.51 [0.43, 0.60]
0.64 [0.52, 0.78]
2.01 [1.59, 2.55]
0.54 [0.44, 0.67]
1.90 [1.38, 2.62]
6.31 [4.48, 8.88]
0.23 [0.15, 0.35]
3.66 × 10−11 a
9.26 × 10−27 b
7.20 × 10−17 b
We have obtained convincing evidence of association between positivity for ZnT8A and the nonsynonymous SNP, rs7522061, in FCRL3, and in our datasets this association was indistinguishable from the association with the non-coding SNP, rs7528684. The T allele of rs7522061, which is associated with positivity for ZnT8A, alters the amino acid sequence (Asp to Asn), which could alter protein function and/or RNA stability/splicing. Only in relation to multiple sclerosis have there been reports of this SNP being associated; the same study also reported disease association with rs7528684 . The minor G allele at rs7528684 is associated with risk of rheumatoid arthritis and systemic lupus erythematosus , protection from autoimmune Addison disease  and multiple sclerosis , and has been found to alter the binding affinity of nuclear factor-κΒ , making it a good functional candidate SNP. Interestingly, FCRL3 is not associated with type 1 diabetes risk , even though it is strongly associated with IA-2A positivity [21, 28]. We have shown here that this association is probably due to a primary association with positivity for ZnT8A and not with IA-2A positivity directly. The association with positivity for ZnT8A was marked equally well by rs7522061 or rs7528684, and consequently, further work is required to unravel the precise nature of the association with the presence of ZnT8A in the FCRL3 region and the mechanisms involved.
None of the regions known to be associated with type 1 diabetes, outside of the HLA, were associated with positivity for ZnT8A. Interestingly, evidence of association with positivity for ZnT8A was confined to the HLA class I region, but the most associated variant, rs9258750, was not associated with type 1 diabetes (p = 0.13 in 8,232 British cases and 9,757 British controls; data not shown). The known associations of IA-2A with HLA-A*24 [14, 32] and of GADA with rs9266722 in HLA class I  were independent of the ZnT8A associations described in the present study. In contrast to previous reports in smaller sample sets, no association with HLA-DQB1*0302, *0604, *04 alleles and ZnT8A was obtained (p > 0.15), neither with positivity for ZnT8RA (p > 0.15) nor ZnT8WA (p > 0.16).
Our study is the largest of its kind to date, using 2,239 case samples, 70% of whom had had ZnT8A measured, having been diagnosed with type 1 diabetes more than 2 years earlier. Therefore, we have been able to confirm recent reports that showed that positivity for ZnT8A declines after diagnosis of disease [12, 13]. Approximately 40% of cases tested for ZnT8A who have had diabetes for 4 years are expected to test positive compared with 62% at diagnosis . In our samples it was comparable (Table 2). A consequence of this decline in positivity for ZnT8A with duration of diabetes is that the rate of ZnT8A negative samples will be elevated amongst cases with longer disease duration. Consequently, almost half the cases classed as ZnT8A negative who had had diabetes for 9 or more years would probably have tested positive had they been tested at diagnosis. This causes the association signal at some loci to be diluted in these samples, but does not affect the validity of the positive associations detected. The HLA and FCRL3 associations with ZnT8A in cases who have had diabetes for ≤2 years are of comparable strength to those who have had diabetes for ≥9 years (ESM Tables 3 and 4). All positive associations were confirmed in the cases from the families affected by type 1 diabetes, all of whom were tested within 2 years of diagnosis. The lack of association outside of the HLA and FCRL3 may be attributable to lack of power, which was exacerbated by the increased frequency of ZnT8A negativity amongst samples with a longer duration of disease. The inverse correlation of positivity for ZnT8A with duration of diabetes was not known when we designed our study, and hence, the statistical power of future studies into the genetic architecture of presence of ZnT8A would benefit from using cases as close to diagnosis as possible, as we have done for the replication in family samples.
For autoantibodies to be causal for type 1 diabetes, the variants associated with risk of type 1 diabetes would also be expected to be associated with autoantibody positivity. Our results, therefore, indicate that autoantibodies to ZnT8 are a downstream event to the primary genetic pathogenesis of type 1 diabetes. Given their specificity for type 1 diabetes , however, this does not detract from their well established value, in combination with other autoantibodies, in assessing risk of the disease [6, 10, 20], but does illustrate how useful biomarkers need not necessarily have associations with genetic markers that confer susceptibility to disease.
We gratefully acknowledge the participation of all the patients, controls and family members.
We thank D. Dunger and B. Widmer of the University of Cambridge, and the British Society for Paediatric Endocrinology and Diabetes for the type 1 diabetes case collection. We also thank P. Clarke, G. Coleman, S. Duley, D. Harrison, S. Hawkins, M. Maisuria, T. Mistry and N. Taylor, all of the University of Cambridge, for preparation of DNA samples.
DNA control samples were prepared and provided by S. Ring, R. Jones, M. and W. McArdle of the University of Bristol, D. Strachan of the University of London and P. Burton of the University of Leicester. We acknowledge use of DNA from The UK Blood Services collection of Common Controls (UKBS collection), funded by Wellcome Trust grant 076113/C/04/Z, by the Wellcome Trust/Juvenile Diabetes Research Foundation grant 061858, and by the National Institute of Health Research of England. The collection was established as part of the WTCCC. This research uses resources provided by the T1DGC, a collaborative clinical study sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Allergy and Infectious Diseases (NIAID), National Human Genome Research Institute (NHGRI), National Institute of Child Health and Human Development (NICHD) and Juvenile Diabetes Research Foundation International (JDRF), and supported by U01 DK062418. We acknowledge bioinformatics assistance from O. Burren of the University of Cambridge. We gratefully acknowledge N. Walker of the University of Cambridge for his contribution to data management.
This work was funded by the Juvenile Diabetes Research Foundation International, the Wellcome Trust and the National Institute for Health Research Cambridge Biomedical Centre. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (079895). The research leading to these results has received funding from the European Union's 7th Framework Programme (FP7/2007-2013) under grant agreement no 241447 (NAIMIT). The collection of DNA for the British 1958 Birth Cohort was funded by the UK Medical Research Council and Wellcome Trust (MRC grant G0000934, WT grant 068545/Z/02).
The study was supported in part by grants from the German Federal Ministry of Education and Research (BMBF) to the Competence Network for Diabetes mellitus (FKZ 01GI0805-07), and from the European Union (EP7-HEALTH-2007, DIAPREPP N202013). PA was supported by JDRF (11-2005-1117). The study was supported in part by grants from the Juvenile Diabetes Research Foundation Autoimmunity Prevention Center, Denver, CO (JDRF #4-2007-1056) and the NIH Diabetes and Endocrine Research Center (NIDDK P30 DK57516).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
JMMH analysed and interpreted the data and wrote the manuscript. SK, AGZ, EB and PA tested case control samples for ZnT8A positivity. HS managed and prepared DNA samples and interpreted data. DJS performed TaqMan genotyping. JW and JH measured ZnT8A in T1DGC families. EB, JAT, PA and JMMH designed the study. All authors reviewed/edited/approved the manuscript.
This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.