The cation efflux transporter ZnT8 (Slc30A8) is
a major autoantigen in human type 1 diabetes
Janet M. Wenzlau, Kirstine Juhl*, Liping Yu, Ong Moua, Suparna A. Sarkar, Peter Gottlieb, Marian Rewers,
George S. Eisenbarth, Jan Jensen, Howard W. Davidson, and John C. Hutton†
Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, PO Box 6511, 1775 North Ursula Street,
Aurora, CO 80045
Edited by Diane Mathis, Harvard Medical School, Boston, MA, and approved September 6, 2007 (received for review June 22, 2007)
Type 1 diabetes (T1D) results from progressive loss of pancreatic
islet mass through autoimmunity targeted at a diverse, yet limited,
series of molecules that are expressed in the pancreatic ? cell.
Identification of these molecular targets provides insight into the
pathogenic process, diagnostic assays, and potential therapeutic
agents. Autoantigen candidates were identified from microarray
expression profiling of human and rodent pancreas and islet cells
and screened with radioimmunoprecipitation assays using new-
onset T1D and prediabetic sera. A high-ranking candidate, the zinc
transporter ZnT8 (Slc30A8), was targeted by autoantibodies in
60–80% of new-onset T1D compared with <2% of controls and
<3% type 2 diabetic and in up to 30% of patients with other
autoimmune disorders with a T1D association. ZnT8 antibodies
(ZnTA) were found in 26% of T1D subjects classified as autoanti-
body-negative on the basis of existing markers [glutamate decar-
boxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), anti-
bodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)].
Individuals followed from birth to T1D showed ZnT8A as early as
2 years of age and increasing levels and prevalence persisting to
disease onset. ZnT8A generally emerged later than GADA and IAA
in prediabetes, although not in a strict order. The combined
detection rates to 98% at disease onset, a level that approaches
that needed to detect prediabetes in a general pediatric popula-
used here will potentially generate other diabetes autoimmunity
markers and is also broadly applicable to other autoimmune
autoantibody ? zinc transport ? prediabetes
selective destruction of the ? cells of the pancreatic islets of
Langerhans (1). In the nonobese diabetic (NOD) mouse, B
lymphocytes also play a role in pathogenesis (2), although their
levels of circulating autoantibodies in man provide important
predictive markers for the underlying autoimmunity that may
precede clinical disease by many years, during which time,
therapeutic intervention may be effective (4–7). The histological
determination of islet cytoplasmic autoantibodies (ICA) (8) or
the combined biochemical measurement of antibodies to insulin
(IAA) (9), the 65-kD form of glutamate decarboxylase (GADA)
(10) and the protein tyrosine phosphatase IA2 (IA2A) (11) can
identify 80% or more of patients at disease onset or at risk of
with a family history of autoimmune diabetes have been instru-
mental in selection of patients for entry into clinical trials (12)
but currently fall short of the high sensitivity and specificity
required for detection of (pre)diabetes in the general population
where the prevalence is of the order of 0.3% even when genetic
susceptibility markers are also included (13).
Recent progress toward the development of immunological-
based therapies for T1D and other autoimmune disorders in
ype 1 diabetes (T1D) is a T cell-dependent tissue-specific
autoimmune disease, characterized in animal models by the
humans (14) highlights the need for development of further
markers of B and T cell autoimmunity in diabetes. These can be
immune intervention is most likely to be effective and may also
be therapeutic agents in their own right. We report the identi-
fication of such a marker based on a multidimensional analysis
of microarray data, subsequent development of sensitive immu-
noprecipitation assays to conformational epitopes in the mole-
cule, and their application to study of humoral autoimmunity in
new-onset and prediabetic patients.
Microarray Analyses. ZnT8 was originally identified as an EST
chosen from a list of 68 candidate islet autoantigens that was
compiled from multidimensional analyses of microarray mRNA
expression profiling experiments. The screening process was
initiated by interrogation of public domain multitissue custom
arrays (Gene Atlas V2; Novartis, http://symatlas.gnf.org) (15)
using three criteria of enrichment of gene expression in islets,
resulting in the acceptance of 300 genes. One hundred sixty of
these were excluded on the basis of their being more abundant
in an ?TC1-6 glucagonoma cell line than ?TC3 insulinoma cell
line or being expressed at comparable levels in mPAC pancreatic
cells. Another 40 were excluded on the basis that they were
expressed at much lower levels in mouse islet and FACS-sorted
?-cells than in the cell lines. The tissue expression of the
remaining 100 genes was evaluated from the frequency with
which ESTs have been sequenced from cDNA libraries from 49
the pancreatic EST frequency relative to the global EST fre-
quency. The multiplication product of these indices was used to
rank the genes [supporting information (SI) Table 1]. Fig. 1
shows two-dimensional maps of pancreatic specificity relative to
the pancreatic EST frequency and islet transcript abundance
determined by microarray analysis.
The final list of 68 candidate genes included major known
diabetes autoantigens, notably insulin (9), GAD65 (GAD2) (10),
putative targets of autoimmunity (heat shock protein 90B (17),
carboxypeptidase E (CPE) (18), islet glucose 6 phosphatase-
Author contributions: J.M.W., G.S.E., H.W.D., and J.C.H. designed research; J.M.W., K.J.,
J.J., H.W.D., and J.C.H. contributed new reagents/analytic tools; J.M.W., K.J., L.Y., S.A.S.,
P.G., M.R., G.S.E., J.J., and J.C.H. analyzed data; and J.C.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: GADA, glutamate decarboxylase antibodies; IAA, antibodies to insulin;
IA2A, IA2 antibodies; ICA, islet cytoplasmic autoantibodies; T1D, type 1 diabetes; ZnTA,
*Present address: Joslin Diabetes Center, 1 Joslin Place, Boston MA 02215.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
October 23, 2007 ?
vol. 104 ?
related protein (IGRP) (19), islet amyloid polypeptide (IAPP)
(20), pancreatitis-associated protein (humanReg3a/Mouse
Reg2) (21), ICA69 (22), imogen 38 (23), peripherin (24), sox13
(25), and GAD67 (GAD1) (26). The appearance of GAD65,
CPE, IA2, and phogrin on the list, although gratifying, was
surprising, given their known association with neuroendocrine
tissues. This was attributed to their high representation in
pancreas cDNA libraries, which probably underestimates their
representation in islet tissue, which represents ?5% of the
Slc30A8 (ZnT8) ranked fourth in the hierarchy of candidate
genes based on its pancreas and islet specificity SI Table 1) and
28th with respect to islet expression level. The islet specificity is
consistent with previous studies based on PCR analysis (27) and
our own quantitative PCR analyses (data not shown). The only
other tissue showing significant expression was testis (seven
clones from 337,730 ESTs), although the frequency of appear-
ance is still 20-fold lower than the whole pancreas. Of the more
abundantly expressed genes in human pancreas, only INS,
GAD2, and IGRP ranked higher, and all of these are prominent
ZnT8 Autoantibodies and Identification of Regional Epitopes. A ra-
dioimmunoprecipitation assay for ZnT8 autoantibodies was
developed by using [35S]methionine-labeled in vitro translation
products of different fragments of human ZnT8 (Fig. 2). Results
with new-onset T1D sera using the 369-aa ORF were encour-
aging (25% sensitivity, 98% specificity); however, a nonspecific
binding of ?5% and unacceptable false-positive rate precluded
its use for patient screening (Fig. 2A). Approximately half of the
ZnT8 structure lies within six membrane-spanning regions (Fig.
2B) that are unlikely to be accessible to antibodies and probably
impede its folding in an aqueous environment. Assays were
therefore developed to a series of fragments of the predicted
luminal and cytosolic regions as either individual segments or
fusion constructs. Autoreactivity to amino acids 1–74 of the N
terminus (Fig. 2A) broadly correlated with that of the ORF
results (R2? 0.605, P ? 0.001, n ? 186), but the assay showed
low sensitivity (8.0% sensitivity, 98% specificity, n ? 223). In
contrast, a C-terminal construct spanning amino acids 268–369
produced a robust and sensitive assay (50% sensitivity, 98%
specificity). A synthetic molecule that combined both N- and
C-terminal sequences in a single-chain construct performed
more reliably than the ORF construct (Fig. 2C), and the levels
of antibodies were correlated (R2? 0.494, P ? 0.001; SI Fig. 7),
suggesting that the transmembrane regions and the short cyto-
plasmic and luminal connecting peptides were not major con-
tributors of ZnT8 autoantibody epitopes. The N/C construct,
however, did not incorporate all of the epitopes recognized by
C-terminal reactive sera (SI Fig. 7). Assays of C-terminal and N-
and C-terminal fusion proteins (N/C) thus complemented one
another, leading to the detection of 63% of diabetic individuals.
the current standard biochemical autoantibodies used to diag-
nose T1D autoimmunity.
of the large cation efflux family (10 mammalian homologues;
almost 100 family members) of which at least 7 are expressed in
islets (27–29) raising the question of whether autoreactivity to
ZnT8 in T1D might be directed at other family members.
Immunoprecipitation assays performed with nine strongly
ZnT8-positive diabetic sera with C-terminal constructs of ZnT3
(Slc30A3) (42% identity) and ZnT5 (Slc30A5) (22% identity)
were negative (data not shown), whereas preincubation of the
same sera with 10 ?g of recombinant His-tagged C-terminal
ZnT8 reduced binding to ZnT8 by 93 ? 2.4% (n ? 7) (data not
Three individuals (37.5%) from a group of eight T1D subjects
who were negative for GADA, IA2A, and IAA but positive for
histological islet cytoplasmic antibody (ICA), showed ZnT8A
(data not shown). Thirty five from a group of 133 (26.3%) young
(mean age 13, range 3–23) insulin-treated patients who were also
t i c i f i c
s s i T
Pancreatic clonal frequency (ppm)
Islet mRNA expression (Ln2 signal)
ESTs) and against the level of mRNA expression in human islets from microar-
ray data (SI Table 1). Approximate positions are shown for diabetes autoan-
tigens (filled symbols).
Identification of candidate autoantigens. Pancreas specificity is
62.3% +ve n=223
63.2% +ve n=223
65.0% +ve n=223
N-termC-term N/C fusion
c ( y t i v i t c
o i d
R e t a t i p i c
e r p
(amino acids 264–369) constructs, and a single-chain fusion of the N and C
termini (B). (C) The overlap in autoantibody prevalence to each construct.
Wenzlau et al.
October 23, 2007 ?
vol. 104 ?
no. 43 ?
ICA-negative were reactive to ZnT8, some quite strongly (Fig.
3). In contrast, only 1 of 30 type 2 diabetes patients had ZnT8A,
probably misclassified because he also had GADA (data not
Autoreactivity to islet cell proteins is often associated with
other tissue-specific immune disorders such as Graves, Addi-
son’s, and celiac disease (30–32). Accordingly, ZnT8A were
disease without symptoms of diabetes (Fig. 3) along with GADA
(7 of 35, 20%), IA2A (4 of 35, 11.4%), and IAA (1 of 19, 5.2%).
Two subjects from a group of 15 who were 21-hydroxylase
antibody-positive but without clinical Addison’s disease were
also ZnT8A-positive (13.3%). Similarly, 12 of 39 (30.8%) non-
diabetic, tissue-transglutaminase autoantibody-positive individ-
uals related to T1D patients with celiac disease showed ZnT8A
(Fig. 3). ZnT8A measurements on a group of 25 systemic lupus
patients with nucleic acid antibodies and a group of 50 rheu-
matoid arthritis patients, however, were negative (data not
Association of ZnT8 with Other T1D Autoantibodies.Given their high
prevalence, ZnT8A obviously overlap with GADA, IA2A, and
IAA at disease onset. Analyzed in terms of the levels of
reactivity, ZnT8 N/C antibodies correlated weakly with IA2A
(R2? 0.095, P ? 0.0001; SI Fig. 7) but not with IAA or GADA.
Given also that ZnT8A can be present in otherwise antibody-
negative individuals (Fig. 3), we conclude that ZnT8 autoimmu-
nity is likely an independent T1D marker. This is also evident
from the association between each of the autoantibody markers
based on prevalence at disease onset (Fig. 4). Individually, IA2,
GAD, INS, and ZnT8 antibodies were detected in 72%, 68%,
55%, and 63% of new-onset patients (n ? 223). The combined
measurement of GADA, IA2A, and IAA, the current gold
standard, raised the detection of autoimmunity to 94%. ZnT8A
measurements, if substituted individually for GADA, IA2A, or
IAA, detected a similar number of diabetic patients; however,
the real value of ZnT8A lies as an fourth measurement (Fig. 4B);
inclusion of the ZnT8 assays reduced the number of diabetic
autoantibody-negative individuals from 5.8% to 1.8% and in-
creased the number who tested positive for two or more auto-
antibodies from 72% to 82% (n ? 223, P ? 0.013). Previous
longitudinal studies indicate that the prevalence of multiple
diabetic autoantibodies is a better index of disease progression
than prevalence or titer of antibodies directed at individual
antigens (4, 5, 33). This may indicate intermolecular epitope
spreading and, hence, worsening autoimmunity, although it
should be noted that seropositivity to two antigens has a higher
specificity (100%) than one (92–98%) simply because control
individuals seldom show two autoantibodies.
Stratification of the results with subject age at diabetes onset
showed that both the prevalence (Fig. 5) of ZnT8 C-terminal and
ZnT8 N/C antibodies were low in younger individuals but
increased dramatically from ?3 yr onwards. Prevalence of ZnTA
peaked at 80% in late adolescence and tended to decline
thereafter (58% in a 23- to 30-yr-old group, n ? 19). ZnT8
C-terminal and ZnT8 N/C reactivity changed in parallel and
to ZnT8A, the prevalence of IAA decreased at older ages, thus
emphasizing the utility of ZnT8 antibodies as an additional
marker in older subjects. The levels as well as the prevalence of
ZnTA increased with age (SI Fig. 8).
Longitudinal Studies of Prediabetic Autoimmunity and Risk Predic-
tion. The question of when ZnT8 autoimmunity emerges in
relation to clinical disease was addressed by using samples from
the Diabetes Autoimmunity Study in Youth (DAISY) (34).
These included 30 first-degree relatives of T1D patients and 13
individuals from the general population selected for high-risk
HLA genotypes (DR3 and/or 4) followed prospectively from ?1
yr of age to diabetes [median age at onset 6.8 yr (1.9–14.3)]. At
onset, 95.3% had antibodies to at least one of the four antigens;
57.9% for ZnT8, 61.9% for GAD, 53.5% for IA2, and 59.1% for
INS, which was consistent with the new-onset data (SI Fig. 8 and
Figs. 4 and 5), considering that this ongoing prospective study is
presently biased toward disease emergence at younger ages.
Life-table analysis of the antibody appearance with time (Fig.
6 A–C) showed that ZnT8A usually preceded disease by many
years and frequently appeared by 3 yr of age. IAA was more
prevalent than ZnT8A between the ages of 1.5–3 yr (?2, P ?
0.035), and a similar trend was observed between ZnT8A and
c ( y t i v i t c
o i d
R e t a t i p i c
e r p
GADA-, IA2A-, IAA-, and ICA-negative new-onset T1D subjects, nondiabetic
subjects who were 21-hydroxylase antibody-positive with or without Addi-
with celiac disease.
Uniquiness of ZnTA. ZnT8 C-terminal autoreactivity was measured in
≥ 2Ab 79.4%
≥1 Ab 92.4%
≥1 Ab 95.5%
≥1 Ab 94.2%
≥1 Ab 96.0%
Seropositive individuals evaluated with three-autoantibody standard or with
C-terminal and N/C assays in the one measurement. (B) Seropositive individ-
uals evaluated with four-autoantibody standard.
www.pnas.org?cgi?doi?10.1073?pnas.0705894104Wenzlau et al.
GADA (?2, P ? 0.134). IA2A, like ZnT8A, was less prevalent
than IAA (?2, P ? 0.002) at early time points and showed a
similar trend relative to GADA (?2, P ? 0.106). The levels of
ZnT8A (SI Fig. 9) were typically low before 2 yr of age but
increased progressively in the following 2 yr. A similar pattern
was followed by IA2, whereas GAD and INS reached a plateau
at 2 yr. The differences between levels recorded at year 3 and
diagnosis were significant in the case of ZnT8A (P ? 0.006,
Mann–Whitney parametric test, n ? 31) and IA2 (P ? 0.034) but
not GAD (P ? 0.636) or INS (P ? 0.769).
The progression of autoreactivity in 10 individuals is illus-
trated in SI Fig. 10. ZnT8A were observed in 17 of the 27 cases
(62.9%) before diabetes, GADA in 19 (70.3%), IA2A in 14
(51.8%), and IAA in 23 (85.1%). The corresponding numbers at
disease onset were 17 (62.9%), 15 (55.5%), 13 (48.1%), and 19
(70.3%), indicating some dropout of GADA and IAA. Individ-
the levels attained, and the sequence in which they appeared.
The median age of appearance of GADA (2.0 yr, range 1.26–
8.87) and IAA (2.2 yr, range 0.8–8.9) tended to be earlier than
that of IA2A (2.7yr, range 1.9–8.7) and ZnT8A (3.6 yr, range
1.3–8.9), concordant with the life-table analysis (Fig. 6 A–C).
However, there was no strict order of appearance of antibodies,
and in two individuals, ZnT8A were the only ones observed. Two
ZnT8A persisted to diagnosis, falling only marginally in six
individuals. Such stability contrasted to GADA and IAA, which
frequently declined with age, often leading to dropout. The
interval separating ZnT8A appearance and disease varied
widely, in two cases, within a year, in others up to 7 yr. In a
pair-wise comparison, diabetes emerged sooner after ZnT8
seropositivity than GADA (4.14 ? 2.62 vs. 5.17 ? 2.85 yr, paired
t test P ? 0.009, n ? 12), sooner than IAA (3.62 ? 2.15 vs. 4.56 ?
2.41 yr, P ? 0.01, n ? 14) but at approximately the same time
relative to IA2A appearance (3.72 ? 2.33 vs. 3.65 ? 1.49 yr, P ?
0.889, n ? 11). These differences reflect the earlier onset of
GADA and IAA relative to ZnT8 (Fig. 6 A and B), and it is
concluded that ZnT8 autoantibodies, although arising later, do
not predict imminent progression to clinical disease any more
than GADA, IA2A, or IAA.
To test whether the determination of ZnT8 autoantibodies
enhances the ability to predict disease in the presence of other
autoantibodies, an analysis was performed on the first serum
samples from each of 88 DAISY subjects that had tested positive
for a single biochemical antibody (GADA, IA2A, or IAA). The
study focused on individuals who were older than 3 when
given to the initial antibody type nor to subsequent emergence
of other autoantibodies. Of this group, 36.8% (7 of 19) of
individuals with ZnT8 antibodies progressed to clinical diabetes
compared with 7.3% (5 of 69) of those without (P ? 0.003,
two-tailed Fisher exact test). Life-table analysis (Fig. 6D) con-
firmed this observation and showed that the presence of ZnT8A
significantly increased the probability that an individual other-
wise classified as low risk would progress to clinical diabetes
within 5 yr (P ? 0.007). A previous prospective study restricted
to IAA, GADA, and IA2A (33) has similarly shown that the risk
of development of diabetes within 5 yr increased from 15% with
one autoantibody to 44% with two.
The ZnT8 transporter was pursued as a candidate diabetes
autoantigen based on a bioinformatic screen of genes aimed at
testing the hypothesis that tissue-specific autoimmunity is often
directed at molecules that are expressed in a tissue-specific
manner at moderate-to-high abundance. It fulfilled another
postulate in being associated with the regulated pathway of
secretion (27), a feature common to many ?-cell and thyroid
autoantigens (35). ZnT8 is thought to be responsible for the
concentration of Zn2?in the granule lumen (27), although this
is currently unproven because the ?-cell expresses other more
widely distributed Slc30A family members such as ZnT2, 4, and
5, some with granule localization (29). We found no evidence
that autoreactivity to ZnT8 was shared by the most-closely
ZnN/C R2=0.751, P=0.003
ZnC R2=0.812, P=0.001
Age at onset (yr)
GAD R2=0.220, P=0.202
Age at onset (yr)
IA2 R2=0.025, P=0.687
e l a
e r P
INS R2=0.826, P=0.001
e l a
e r P
subjects ranging from ?1 yr to 18 yr old were grouped in 2-yr intervals to
relation one another and to GADA, IA2A, and IAA.
Relationship of ZnT8A to age at diabetes onset. New-onset T1D
Follow up (yr)
69 49 32 17 7 2
19 16 11 6 4 1
27 24 15 12 9 4
27 26 14 8 3 1
27 18 12 9 5 3
27 26 14 8 3 1
26 16 8 6 4 3
27 26 14 8 3 1
( c i t e
a i d n
d i c
n I e
v i t a l u
d i c
n I e
v i t a l u
d i c
n I e
v i t a l u
bodies in 27 subjects followed to T1D is shown for ZnT C-terminal autoreac-
tivity relative to GADA, IA2A, and IAA. (D) ZnT8 C-terminal autoantibodies
were determined on the first serum samples to test positive for GADA, IA2, or
each time point are indicated.
ZnT8A in prediabetes. (A–C) The cumulative incidence of autoanti-
Wenzlau et al.
October 23, 2007 ?
vol. 104 ?
no. 43 ?
related family members and, in general terms, pointed to
epitopes being confined to ZnT8. A recent genome-wide asso-
ciation study demonstrated an association of a series of ZnT8
polymorphisms with human type 2 diabetes (36). The chromo-
been implicated in human T1D.
ZnT8, like another diabetes autoantigen, IGRP (19, 37), is a
multispanning transmembrane protein that presents a challenge
to development of assays for humoral and cell-mediated auto-
reactivity because of the difficulty of maintaining its tertiary
structure outside of a membrane environment. Sensitive and
specific ZnT8A assays nevertheless were developed by using
radioimmunoprecipitation of in vitro-translated products gener-
ated from soluble domains of the protein and construction of
single-chain molecules fusing C- and N-terminal domains. Most
structural prediction models place both the C and N termini in
the cytoplasm and are thus unlikely to be exposed on the cell
surface during granule exocytosis. A similar topology for three
other membrane-associated diabetes autoantigens, GAD65,
IA2, and phogrin, has been inferred, leading to speculation that
these antigens might encounter immune surveillance only as
alternatively spliced forms (38, 39), after apoptosis of ?-cells
early in postnatal life (40) or after immune-mediated destruction
of islets targeted at other cell-surface or secreted autoantigens.
As a protein that is initially incorporated into the endoplasmic
reticulum (ER), it might also be delivered to the MHC process-
ing compartment by premature termination of translation prod-
ucts or after ER stress (35). The observation that high levels of
ZnT8A emerged late relative to IAA and GADA fits the idea
that ZnT8 antigen presentation occurs secondary to immune
damage. However, the absence of hierarchy of autoantibody
either the initiating antigen is unknown or that there exist
multiple pathways and diverse forms of human T1D from an
immunological perspective. The complex kinetic series might
by different environmental factors.
ZnT8A were persistent in the prediabetic phase and proved a
useful independent marker of autoimmunity either alone in
antibody-negative subjects or in conjunction with IAA, GADA,
or IA2A, where it increases the likelihood of detecting a
prognostic second autoantibody (6). ZnT8A should be especially
useful in older individuals in whom insulin autoantibodies wane
with age. Unlike GAD and IA2, ZnT8 is highly ?-cell-specific,
and thus ZnT8A measurements may be useful in monitoring islet
destruction after onset and in evaluating therapeutic interven-
tions that limit ?-cell-specific autoreactivity or restore ?-cell
mass. IAA measurements in these circumstances are precluded
because insulin administration itself induces insulin antibodies.
The bioinformatics approach that identified ZnT8 as a can-
didate autoantigen can potentially be extended to other tissue-
specific autoimmune disorders. In the case of T1D, with a
prevalence of 0.3% in the Caucasian population, the develop-
ment of further robust assays for humoral autoreactivity in
combination with genetic screening raises the potential of pe-
diatric screening for T1D susceptibility in the general popula-
of the autoantigen epitopes are conformational, requiring struc-
tural evaluation and molecular engineering strategies to develop
Human islets of 75–80% purity and 76–96% viability were
obtained from the National Institutes of Health Islet Cell
Resource (ICR) program. Total RNA extracted with TRIzol
reagent was quantified (Agilent Bioanalyzer; Agilent Technol-
ogies, Palo Alto, CA) and processed for hybridization to Af-
fymetrix U133A Ver 2.0 oligonucleotide microarrays by using
standard procedures (Affymetrix, Santa Clara, CA). Results
from five different islet preparations were normalized by the GC
robust multi-array average (GCRMA) procedure.
The Novartis custom oligonucleotide array of 79 human
criteria: fold enrichment of genes in islets versus whole pancreas,
fold enrichment in islets versus median expression on the array,
and tissue specificity based on the calculation of Shannon
entropy (41). A compilation of 300 genes was then compared
with the corresponding mouse genes on MOE40 microarrays of
pancreatic tumor cell lines (?TC1–6 glucagonoma, ?TC3 insu-
linoma, and mPAC ductal cell lines) to filter out housekeeping
genes and genes expressed at higher levels (?2.5-fold) in non-?
islet cells. The 140 genes that passed these criteria were evalu-
ated by the Unigene EST profile viewer in 49 normal human
tissues based on the frequency with which ESTs have been
observed in cDNA libraries. The pancreatic abundance (pan-
creatic clones per million cDNAs) and pancreatic specificity
(pancreatic clonal frequency per global frequency) were calcu-
lated and the product used as a score to rank the genes (SI Table
1). The data and programs used in these analyses can be accessed
through http://genespeed.uchsc.edu/development?1, a public do-
main database geared to transcriptional and cell biological
analyses focused on pancreatic islet development (42).
The human ZnT8 (Slc30A8) ORF (ORF) was amplified by
PCR from human islet cDNA prepared with an iScript kit
(Bio-Rad, Hercules CA) using the primers; forward FL-
CACCATGGAGTTTCTTGAAAG and reverse FL-CTAGT-
CACAGGGGTCTTCAC. An N-terminal construct used the
same forward primer and the N-terminal reverse primer
GAGTTTCCCACTTGGCATAGGC and a C-terminal con-
struct the C-forward CACCATGAAGGACTTCTCCATCT-
TACTC and the reverse FL primer. The N/C construct was made
by combining a N-terminal PCR product generated with the
forward FL primers and a linker N/C reverse primer (GTAA-
TTCCACTTGGC) and a C-terminal PCR product generated
with a N/C forward primer (GCCAAGTGGAAACTCTGT-
TCTGGTGGCGGAAAGGACTTCTCCATCTTAC) and re-
verse FL primer. The products were gel purified, mixed and
reamplified with the FL primer set to generate a single-chain
N/C fusion sequence with a AKWKLCSGGGKDFSIL junction.
All constructs were cloned into pCDNA3.1 directional TOPO
vector (kit 45–0158; Invitrogen, Carlsbad, CA), and sequence
Plasmid DNA (2 ?g) was incubated in a 100 ?l of an in
vitro-coupled transcription/translation reticulocyte lysate reac-
tion (TNTQuick T7 promoter; Promega, San Luis Osbispo, CA)
with 20 ?Ci (1 Ci ? 37 GBq) of [35S]methionine (1,000 Ci/mmol;
Amersham Bioscience, Pittsburgh, PA) and the products gel-
filtered on G25 Sephadex (NAP5 column; GE Healthcare,
Piscataway, NJ). Radioactivity incorporated into protein was
determined by precipitation with 5% (wt/vol) trichloroacetic
acid after alkaline hydrolysis of any [35S]Met tRNA left in the
sample with 1M NaOH (percent incorporation: C-terminal
10.0 ? 2.1; N/C 10.6 ? 0.4). Human serum samples (5 ?l) were
incubated overnight at 4°C with 25,000 cpm of the above
translation product in 50 ?l of PBS containing 0.1%(wt/vol)
BSA, 0.15% Nonidet P-40, and 0.02% NaN3in a 96-well plate.
Immobilized Protein A (10 ?l of 50% (vol/vol) slurry (Protein A
Sepharose 4 Fast Flow 17–5280; Amersham Bioscience) was
added to each well, and 45 min later, the beads were recovered
by filtration (MADVNOB50 96-well plates; Millipore, Billerica,
MA) and bound radioactivity determined by liquid scintillation
counting (Wallac 1450 Microbeta Trilux counter; PerkinElmer,
Waltham, MA). GADA IA2A and IAA assays followed similar
standard protocols (43).
www.pnas.org?cgi?doi?10.1073?pnas.0705894104Wenzlau et al.
Each ZnT8A assay was run with 16 matched control samples Download full-text
and an eight-step doubling dilution of a high-titer T1D sera
standard prepared from a pool of nine new-onset sera, which at
a 1:10 dilution, precipitated 102.5 ? 2.3% (n ? 25) of the added
radioactivity. Half-maximal binding occurred at a 1:50 dilution.
In the standard C-terminal assay, blanks (no sera) averaged
150 ? 26 (seven experiments) and control sera 259 ? 58 cpm (22
experiments). Intrassay coefficient of variation (CV) on controls
ranged from 8–16% (n ? 16) and interassay CV 21% (n ? 30).
Cutoff values were calculated as the mean ? 4 SD of the
within-assay controls and globally relative to 368 DAISY control
sera at a 98% cutoff. Normalization was achieved by using the
immunoprecipitation index (sample ? control)/(high stan-
dard ? control) that gave a cutoff value ? 0.010–0.012. In the
case of the longitudinal study (SI Fig. 10), results were expressed
as the log10fold SD to enable compare IAA data from assays
with different formats. The ZnT N/C assay blanks were 215 ? 40
cpm (n ? 16), control values 370 ? 40 cpm (n ? 268) and 99th
percentile cutoff index (0.015). The C-terminal assay was vali-
dated against blinded sera from 50 newly diagnosed diabetic
patients (aged 18–28) and 100 age-matched controls provided by
the Diabetes Autoantigen Standardization Program (DASP)
within the Centers for Disease Control (44). The assay showed
a 58% sensitivity at 100% specificity and 64% sensitivity at 95%
Subjects were drawn from newly diagnosed T1D patients at
the Barbara Davis Center, their relatives, and from DAISY
participants. The new-onset populations were comprised as
follows: T1D n ? 277; mean age 12.1 (range 1–46), 87%
Caucasian, 6.3% Hispanic; Control n ? 150; mean age 13.1
(range 1–55), 72% Caucasian, 15.1% Hispanic; T1D but all
Ab-negative n ? 133: mean age 13.0 (3–23), 71% Caucasian
mean age 38.7 (range 16–72); Addison’s n ? 15 mean age 33.4
(range 6–79) 100% Caucasian; nondiabetic tTG Ab-positive
first-degree relatives of tTG T1D n ? 39, mean age 12.2 (4–21)
100% Caucasian. Patients from the DAISY cohort were selected
in the following groups: First positive antibody study n ? 88:
mean age 4.5 (range 3–14), 77% Caucasian 13.0% Hispanic;
DAISY followed to diabetes 43 subjects, 441 samples: median
age 4.9 (range 0.6–11), 100% Caucasian; and DAISY controls
n ? 268: mean age 6.8 (range 1.2–46), 66.5% Caucasian. The
latter were healthy volunteers, the parents and children in the
newborn cohort of the DAISY study, and parents of the DAISY
the groups ranged from 0.8 to 1.4. Informed consent was
obtained from participants and/or parents under approved In-
stitutional Review Board oversight. Statistical analyses were
performed with the Prism 4 software package www.graphpad.
com. Results are expressed as mean ? SD unless otherwise
We thank Kathy Barriga, Jennifer Barker, Michael Holers, and Teri Aly
for their advice and assistance in retrieving archived serum samples. This
work was supported by the Children’s Diabetes Foundation in Denver,
CO, the University of Colorado Health Sciences Center Diabetes and
Endocrinology Research Center [National Institutes of Health (NIH)
Grant P30 DK57516], the Beta Cell Biology Consortium (NIH Grant
U19 DK61248), and the Juvenile Diabetes Research Foundation.
1. Anderson MS, Bluestone JA (2005) Annu Rev Immunol 23:447–485.
2. Silveira PA, Serreze DV, Grey ST (2007) Front Biosci 12:2183–2193.
3. Martin S, Wolf-Eichbaum D, Duinkerken G, Scherbaum WA, Kolb H,
Noordzij JG, Roep BO (2001) N Engl J Med 345:1036–1040.
4. Bingley PJ, Christie MR, Bonifacio E, Bonfanti R, Shattock M, Fonte MT,
Bottazzo GF, Gale EA (1994) Diabetes 43:1304–1310.
5. Pihoker C, Gilliam LK, Hampe CS, Lernmark A (2005) Diabetes 54 (Suppl
6. Verge CF, Gianani R, Kawasaki E, Yu L, Pietropaolo M, Jackson RA, Chase
HP, Eisenbarth GS (1996) Diabetes 45:926–933.
7. Achenbach P, Warncke K, Reiter J, Williams AJ, Ziegler AG, Bingley PJ,
Bonifacio E (2006) Diabetologia 49:2969–2976.
8. Bergerot I, Souchier C, Thivolet C (1993) Can R Acad Sci III 316:1368–1373.
9. Palmer JP, Asplin CM, Clemons P, Lyen K, Tatpati O, Raghu PK, Paquette TL
(1983) Science 222:1337–1339.
10. Baekkeskov S, Aanstoot HJ, Christgau S, Reetz A, Solimena M, Cascalho M,
Folli F, Richter-Olesen H, De Camilli P (1990) Nature 347:151–156.
11. Bonifacio E, Lampasona V, Genovese S, Ferrari M, Bosi E (1995) J Immunol
12. Schatz DA, Bingley PJ (2001) J Pediatr Endocrinol Metab 14 (Suppl 1):619–622.
13. Hermann R, Bartsocas CS, Soltesz G, Vazeou A, Paschou P, Bozas E,
Malamitsi-Puchner A, Simell O, Knip M, Ilonen J (2004) Diabetes Metab Res
14. Chatenoud L (2006) Int Rev Immunol 25:215–233.
15. Su AI, Cooke MP, Ching KA, Hakak Y, Walker JR, Wiltshire T, Orth AP,
Vega RG, Sapinoso LM, Moqrich A, Patapoutian A, Hampton GM, Schultz
PG, Hogenesch JB (2002) Proc Natl Acad Sci USA 99:4465–4470.
16. Hawkes CJ, Wasmeier C, Christie MR, Hutton JC (1996) Diabetes 45:1187–
17. Qin HY, Mahon JL, Atkinson MA, Chaturvedi P, Lee-Chan E, Singh B (2003)
J Autoimmun 20:237–245.
18. Castano L, Russo E, Zhou L, Lipes MA, Eisenbarth GS (1991) J Clin
Endocrinol Metab 73:1197–1201.
19. Lieberman SM, Evans AM, Han B, Takaki T, Vinnitskaya Y, Caldwell JA,
Serreze DV, Shabanowitz J, Hunt DF, Nathenson SG, et al. (2003) Proc Natl
Acad Sci USA 100:8384–8388.
20. Clark A, Yon SM, de Koning EJ, Holman RR (1991) Diabet Med 8:668–673.
21. Gurr W, Shaw M, Li Y, Sherwin R (2007) Diabetes 56:34–40.
22. Pietropaolo M, Castano L, Babu S, Buelow R, Kuo YL, Martin S, Martin A,
Powers AC, Prochazka M, Naggert J, et al. (1993) J Clin Invest 92:359–371.
23. Arden SD, Roep BO, Neophytou PI, Usac EF, Duinkerken G, de Vries RR,
Hutton JC (1996) J Clin Invest 97:551–561.
24. Boitard C, Villa MC, Becourt C, Gia HP, Huc C, Sempe P, Portier MM, Bach
JF (1992) Proc Natl Acad Sci USA 89:172–176.
25. Rabin DU, Pleasic SM, Palmer-Crocker R, Shapiro JA (1992) Diabetes
26. Luhder F, Schlosser M, Mauch L, Haubruck H, Rjasanowski I, Michaelis D,
Kohnert KD, Ziegler M (1994) Autoimmunity 19:71–80.
27. Chimienti F, Devergnas S, Favier A, Seve M (2004) Diabetes 53:2330–2337.
28. Clifford KS, MacDonald MJ (2000) Diabetes Res Clin Pract 49:77–85.
29. Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N, Sasaki
R, Mori K, Iwanaga T, Nagao M (2002) J Biol Chem 277:19049–19055.
30. Pietropaolo M, Peakman M, Pietropaolo SL, Zanone MM, Foley TP, Jr,
Becker DJ, Trucco M (1998) J Autoimmun 11:1–10.
31. Barker JM, Ide A, Hostetler C, Yu L, Miao D, Fain PR, Eisenbarth GS,
Gottlieb PA (2005) J Clin Endocrinol Metab 90:128–134.
32. Bao F, Yu L, Babu S, Wang T, Hoffenberg EJ, Rewers M, Eisenbarth GS
(1999) J Autoimmun 13:143–148.
GS (1996) J Autoimmun 9:379–383.
34. Rewers M, Norris JM, Eisenbarth GS, Erlich HA, Beaty B, Klingensmith G,
Hoffman M, Yu L, Bugawan TL, Blair A, et al. (1996) J Autoimmun 9:405–410.
35. Lieberman SM, DiLorenzo TP (2003) Tissue Antigens 62:359–377.
36. Sladek R, Rocheleau G, Rung J, Dina C, Shen L, Serre D, Boutin P, Vincent
D, Belisle A, Hadjadj S, et al. (2007) Nature 445:881–885.
37. Arden SD, Zahn T, Steegers S, Webb S, Bergman B, O’Brien RM, Hutton JC
(1999) Diabetes 48:531–542.
38. Park YS, Kawasaki E, Kelemen K, Yu L, Schiller MR, Rewers M, Mizuta M,
Eisenbarth GS, Hutton JC (2000) Diabetologia 43:1293–1301.
39. Ng B, Yang F, Huston DP, Yan Y, Yang Y, Xiong Z, Peterson LE, Wang H,
Yang XF (2004) J Allergy Clin Immunol 114:1463–1470.
40. Trudeau JD, Dutz JP, Arany E, Hill DJ, Fieldus WE, Finegood DT (2000)
41. Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, Stoeckert CJ, Jr
(2005) Genome Biol 6:R33.
42. Kutchma A, Quayum N, Jensen J (2007) Nucleic Acids Res 35:D674–D679.
43. Wang J, Miao D, Babu S, Yu J, Barker J, Klingensmith G, Rewers M,
Eisenbarth GS, Yu L (2007) J Clin Endocrinol Metab 92:88–92.
44. Bingley PJ, Bonifacio E, Mueller PW (2003) Diabetes 52:1128–1136.
Wenzlau et al.
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no. 43 ?