A Common Nonsynonymous Single Nucleotide
Polymorphism in the SLC30A8 Gene Determines ZnT8
Autoantibody Specificity in Type 1 Diabetes
Janet M. Wenzlau,1Yu Liu,2Liping Yu,1Ong Moua,1Kimberly T. Fowler,1
Sampathkumar Rangasamy,1Jay Walters,1George S. Eisenbarth,1Howard W. Davidson,1
and John C. Hutton1
OBJECTIVE—Zinc transporter eight (SLC30A8) is a major
target of autoimmunity in human type 1A diabetes and is
implicated in type 2 diabetes in genome-wide association studies.
The type 2 diabetes nonsynonymous single nucleotide polymor-
phism (SNP) affecting aa325lies within the region of highest ZnT8
autoantibody (ZnT8A) binding, prompting an investigation of its
relationship to type 1 diabetes.
RESEARCH DESIGN AND METHODS—ZnT8A radioimmuno-
precipitation assays were performed in 421 new-onset type 1
diabetic Caucasians using COOH-terminal constructs incorporat-
ing the known human aa325variants (Trp, Arg, and Gln). Geno-
types were determined by PCR-based SNP analysis.
RESULTS—Sera from 224 subjects (53%) were reactive to
Arg325probes, from 185 (44%) to Trp325probes, and from 142
(34%) to Gln325probes. Sixty subjects reacted only with Arg325
constructs, 31 with Trp325only, and 1 with Gln325only. The
restriction to either Arg325or Trp325corresponded with inheri-
tance of the respective C- or T-alleles. A strong gene dosage
effect was also evident because both Arg- and Trp-restricted
ZnT8As were less prevalent in heterozygous than homozygous
individuals. The SLC30A8 SNP allele frequency (75% C and 25%
T) varied little with age of type 1 diabetes onset or the presence
of other autoantibodies.
CONCLUSIONS—The finding that diabetes autoimmunity can
be defined by a single polymorphic residue has not previously
been documented. It argues against ZnT8 autoimmunity arising
from molecular mimicry and suggests a mechanistic link be-
tween the two major forms of diabetes. It has implications for
antigen-based therapeutic interventions because the response to
ZnT8 administration could be protective or immunogenic de-
pending on an individual’s genotype. Diabetes 57:2693–2697,
expression (1). Susceptibility to the disease is associated
with multiple genetic loci, most prominently HLA alleles
encoding particular major histocompatibility complex
class II glycoproteins (2).
ZnT8 is a newly discovered target of type 1 diabetes
autoimmunity (3) localized to the insulin granule of the
pancreatic ?-cell. It is encoded by SLC30A8, one of nine
human genes for multispanning transmembrane proteins
facilitating Zn2?efflux from the cell and sequestration into
intracellular compartments (4,5). Recent genome-wide as-
sociation studies demonstrate association of ZnT8 gene
polymorphisms with human type 2 diabetes (6–9), notably
a nonsynonymous SNP encoding either Arg or Trp at aa325.
The major, Arg325-encoding C-allele confers a minor risk
(odds ratio 1.07–1.18) of disease. In nondiabetic subjects
with a family history of type 2 diabetes, the C-allele was
associated with increased insulin sensitivity (10), in-
creased circulating proinsulin-to-insulin ratio (11), and
decreased insulin responses in intravenous glucose toler-
ance tests (12), indicating a dominant effect on insulin
secretion, ?-cell mass, or both.
We report here that the type 1 diabetes autoimmune
response to ZnT8 is focused on a few key epitopes, two of
which are defined by the polymorphic aa325residue. To
our knowledge, this is the first reported instance where a
polymorphic variant determines the specificity of the
autoimmune response. It indicates that the autoreactive
B-lymphocyte repertoire is restricted to a few ZnT8
epitopes and is truly self-reactive as opposed to arising as
a bystander response to a foreign antigen.
uman type 1A diabetes results from auto-
immune destruction of pancreatic ?-cells tar-
geted at a restricted number of autoantigens,
many of which show high ?-cell specificity of
RESEARCH DESIGN AND METHODS
Serum samples (n ? 421) were obtained within 2 weeks of type 1 diabetes
diagnosis from patients attending the Barbara Davis Center (median age 11.3
years [range 0.6–58]), 87% Caucasian, and 6.3% Hispanic). The 150 control
subjects (median age 13.1 years [1–55]), 72% Caucasian, and 15.1% Hispanic)
were parents and children in the Diabetes Autoimmunity Study in the Young
(DAISY) general population cohort and parents of the sibling/offspring cohort
(13). The male-to-female sex ratio in both groups was 0.8. Informed consent
was obtained under approved institutional review board oversight.
Genomic DNA was extracted from heparinized blood from 352 of the above
type 1 diabetes patients using standard procedures. Polymorphic variations in
the SLC30A8 gene were determined by qPCR using Taqman probes and an
ABI7000 (ABI, Waltham, MA) targeting the nonsynonymous SNPs rs13266634,
rs2466295 in the 3? untranslated region, and rs6469675 in intron 2. Ascertain-
ment rates were ?99%.
Colorado Denver, Aurora, Colorado; and the2Department of Endocrinol-
ogy, Nanjing Medical University, First Affiliated Hospital, Nanjing, China.
Corresponding author: John C. Hutton, firstname.lastname@example.org.
Received 2 February 2008 and accepted 24 June 2008.
Published ahead of print at http://diabetes.diabetesjournals.org on 30 June
2008. DOI: 10.2337/db08-0522.
© 2008 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Barbara Davis Center for Childhood Diabetes, University of
DIABETES, VOL. 57, OCTOBER 20082693