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The Journal of Clinical Endocrinology & Metabolism 89(5):2222–2227
Copyright © 2004 by The Endocrine Society
doi: 10.1210/jc.2003-031360
Autoimmunity to Islet Proteins in Children Diagnosed
with New-Onset Diabetes
BARBARA M. BROOKS-WORRELL, CARLA J. GREENBAUM, JERRY P. PALMER,
CATHERINE PIHOKER
AND
Departments of Medicine (B.M.B.-W., J.P.P.) and Pediatrics (C.P.), University of Washington, Seattle, Washington 98195;
Department of Medicine (B.M.B.-W., J.P.P.), Department of Veterans Affairs Puget Sound Health Care System, Seattle,
Washington 98108; Benaroya Research Institute at Virginia Mason (C.J.G.); and Children’s Hospital and Regional Medical
Center (C.P.), Seattle, Washington 98105
Autoantibody and T cells reacting with islet proteins have
been demonstrated in patients with type 1 diabetes. In recent
years an increasing number of children have been clinically
classified with type 2 (not ketosis prone, evidence of insulin
resistance, presence of acanthosis nigricans, and obesity) or
indeterminant diabetes (admixture of clinical features of
types 1 and 2). In this study, we compared the islet cell autoantibody and T-cell responses to islet proteins in type 2 (n ⴝ
19) and indeterminant (n ⴝ 16) children (<18 yr of age) to
classic type 1 (n ⴝ 37) diabetic patients. We observed that 37
of 37 type 1 diabetic children demonstrated autoantibody
and/or T-cell reactivity to islet proteins. Fourteen of the 19
C
HILDREN DIAGNOSED WITH diabetes are very commonly presumed to have autoimmune type 1 diabetes.
In clinical practice, physicians use their assessment of phenotypic signs and symptoms to determine whether a child
has type 1 or type 2 diabetes. These include factors such as
age at onset, apparent abruptness of onset of hyperglycemic
symptoms, presence of diabetic ketoacidosis, degree of obesity, presence of acanthosis nigricans, weight loss, and the
perceived need for insulin replacement. Health care providers and researchers (1– 6) have been reporting an increased
prevalence of type 2 diabetes among children largely based
on these clinical phenotypes. However, an alternative hypothesis is that many of these children still have type 1
diabetes, a disease caused by immune-mediated destruction
of the pancreatic ␤-cell but that the phenotype is changing
with the marked increase in childhood obesity.
Type 1 diabetes is characterized by T-cell and autoantibody responses to islet proteins (7–9), whereas type 2 diabetes
is not autoimmune (10). Our laboratory has been interested in
studying the autoantibodies and T-cell autoreactivity to human
islet proteins in type 1 diabetes patients. We previously demonstrated that patients diagnosed with classic type 1 diabetes
between 7 and 35 yr of age have T cells responsive to numerous
islet proteins within 1 yr of diagnosis, whereas normal controls
Abbreviations: DKA, Diabetic ketoacidosis; GAD65Ab, glutamic acid
decarboxylase 65-kDa autoantibody; HLA, human leukocyte antigen;
IA-2, insulinoma associated protein-2 autoantibody; IAA, insulin autoantibody; ICA, islet cell antibody; PBMC, peripheral blood mononuclear
cell; SI, stimulation index.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.
type 2 patients were positive for islet cell autoantibodies, and
six of 14 were positive for T-cell responses to islet proteins. For
the indeterminant patients, 11 of 16 of the patients were positive for autoantibodies, and six of 16 patients were positive
for T-cell responses to islet proteins. These results demonstrate that autoimmunity to islet proteins is present in a high
percentage of children classified as indeterminant or type 2
diabetes. Moreover, the presence of obesity or acanthosis nigracans does not reliably distinguish children with or without islet cell autoimmunity. (J Clin Endocrinol Metab 89:
2222–2227, 2004)
do not (8, 11, 12). Moreover, classic (autoantibody negative)
adult type 2 diabetes patients do not demonstrate T-cell reactivity to islet proteins (11). However, there is a subgroup of
adult patients clinically classified as type 2 diabetes who are
positive for autoantibodies to islet proteins. We previously
demonstrated that these patients commonly also have T-cell
responses to islet proteins (11).
In this study, we asked whether children clinically classified as having type 2 (not ketosis prone, evidence of insulin
resistance, presence of acanthosis nigricans, and obesity, n ⫽
19) or indeterminant diabetes (admixture of types 1 and 2
clinical features, n ⫽ 16) had autoantibodies to islet proteins.
We also examined these patients for T cells reactive to islet
proteins and compared their autoantibody and T cell response with those of classic type 1 diabetes patients (ketosis
prone, insulin requiring, not obese, n ⫽ 37). Moreover, we
asked whether the presence of obesity, urinary ketones, ketoacidosis, or acanthosis nigracans would distinguish children with or without islet cell autoimmunity.
Subjects and Methods
Experimental subjects
Peripheral blood was collected from subjects within 1 month of diagnosis. Assays for autoantibodies, human leukocyte antigen (HLA),
and T-cell assays were performed on the sample. These studies were
approved by the Institutional Review Boards at Children’s Hospital and
Regional Medical Center and the University of Washington. The first 72
newly diagnosed subjects who agreed to participate in the study were
clinically classified by their treating physician as type 1, type 2, or
indeterminant. All nonobese (⬍85th percentile for age and gender) subjects presenting with diabetic ketoacidosis (DKA) were classified as
having classic type 1 diabetes (n ⫽ 37). Type 2 subjects (n ⫽ 19) demonstrated obesity and/or acanthosis nigricans and were negative for
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J Clin Endocrinol Metab, May 2004, 89(5):2222–2227
2223
DKA. Indeterminant subjects (n ⫽ 16) had characteristics that overlapped both types 1 and 2 diabetes. The clinical characteristics, demographic information, and ethnicity of these subjects are shown in Tables
1 and 2.
TABLE 2. Demographic information for patients
Classification
Type 1
diabetes
(n ⫽ 37) (%)
Type 2
diabetes
(n ⫽ 19) (%)
Indeterminant
diabetes
(n ⫽ 16) (%)
Islet cell antibody (ICA) assay
Male
Female
Caucasian
Hispanic
Asian
African-American
Other
65
35
76
8
8
3
5
53
42
32
21
11
11
26
38
63
69
6
13
13
0
Cryostat sections (4 ␮m thick) of human, blood group O pancreas
were adhered to dichromic acid-washed slides coated with poly-l-lysine
hydrobromide. Test sera, preabsorbed with rat liver powder (Sigma
Chemical Co., St. Louis, MO), at dilutions of 1:4 and 1:16, were applied
to slides and the slides incubated overnight in a covered moist chamber
at 4 C. After rinsing with PBS, the appropriate dilution of goat antihuman IgG conjugated with FITC was applied and incubated overnight at
room temperature in a moist chamber. The slides were rinsed with PBS
and a coverslip applied using DABCO mounting media (Sigma). Slides
were coded, randomly arranged, and read in a masked fashion using a
episcopic fluorescent microscope (Nikon Instruments Inc., Melville,
NY). Each section was scored before breaking the code. Our laboratory
has participated in a total of eight International Diabetes Society Workshops and the International Diabetes Society (IDS)-sponsored proficiency programs for ICA with an average sensitivity of 80% and a
specificity of 100% each time. In the IDS-sponsored Combined Antibody
Workshop, our ICA assay had a specificity of 98% and a sensitivity of
76%. Our ICA assay has been validated in a serum exchange with the
Diabetes Prevention Trial-Type 1 ICA core laboratory. In this exchange,
the sensitivity of our assay was 85% with a specificity of 100%. The lower
detection limit for our ICA assay is 1 Juvenile Diabetes Foundation (JDF)
unit and values above the 99th percentile established from approximately 4000 normal school children (4 JDF units) were considered
positive.
standard serum and control sera as in the GAD65 Ab assay were used
to calculate the IA-2 Ab index for each sample. An IA-2 index of 0.017
or more, the 99th percentile based on 200 normal controls, is taken as the
cut-off for positivity. The performance of the assays is monitored by a
set of quality controls and participation in external laboratory proficiency comparison. The laboratory has participated in the DASP. In the
2001 evaluation, the laboratory scored 54% sensitivity and 96% specificity (14). In the 2002 evaluation, the laboratory scored 62% sensitivity
and 89% specificity in the DASP program.
Glutamic acid decarboxylase 65-kDa autoantibody
(GAD65Ab) assay
Cellular immunoblotting
The determination of GAD65Ab levels was performed at the Immunoassay Core of the Diabetes Endocrinology Research Center, University of Washington. GAD65Abs were measured in a radiobinding immunoassay on coded serum samples as described (13). The levels of
GAD65Ab were expressed as a relative index (GAD65 index) using one
positive serum (JDF world standard for ICA) and three negative standard sera from healthy subjects. GAD65 index was calculated and a
positive was considered at 0.085 or less, which is the 99th percentile
based on 200 normal controls. The positive and negative controls are run
in duplicate in each assay. The performance of the assays is monitored
by a set of quality controls and participation in external laboratory
proficiency comparison. The laboratory has participated in the Diabetes
Antibody Standardization Program (DASP). In the 2001 evaluation, the
laboratory scored 76% sensitivity and 94% specificity (14). In the 2002
evaluation, the laboratory scored 84% sensitivity and 96% specificity in
the DASP program.
Insulinoma associated protein-2 autoantibody (IA-2) assay
Autoantibodies to IA-2 were measured under identical conditions as
described for GAD65Ab (13). The plasmid containing the cDNA coding
for the cytoplasmic portion of IA-2 was kindly donated by Dr. G. Eisenbarth (Barbara Davis Research Center, Denver, CO). The same JDF
TABLE 1. Percentages of patients demonstrating different
clinical characteristics
Clinical characteristics
Patient groups
Obesity
(%)
Urinary
ketones (%)a,b
DKA
(%)
AN
(%)
Weight
loss (%)
Type 1 (n ⫽ 37)
Type 2 (n ⫽ 19)
Indeterminant
(n ⫽ 16)
11
95
31
78
26
56
19
0
19
3
100
19
81
32
31
DKA, Diabetic ketoacidosis; AN, acanthosis nigracans.
Differences between type 1 and type 2 patients, P ⬍ 0.05.
Differences between type 2 and indeterminant patients, P ⬍ 0.05.
a
b
Insulin autoantibody (IAA) assay
The IAA assay is performed as previously described (11, 12). The
threshold for positivity was also the 99th percentile of values in normal
controls. IAAs were evaluated before the start of insulin therapy. We
participated in the International Diabetes Society Workshops- and IDSsponsored workshops and proficiency programs for IAA with a sensitivity of 100% and specificity of 100%.
Cellular immunoblotting was performed as previously described for
islets (8, 11, 12, 15) and nonislet proteins (16 –18). Human pancreata were
obtained by University of Washington transplant surgeons, and pancreatic islets were isolated within 48 h, post mortem, at the Islet Satellite
of the Tissue Culture Core of the Diabetes Endocrinology Research
Center (University of Washington, Seattle, WA). Islet cells and tetanus
toxoid were subjected to preparative 10% SDS-PAGE (19). After electrophoresis, the gels were electroblotted onto nitrocellulose (Bio-Rad
Laboratories, Richmond, CA) at 30 mA overnight, nitrocellulose particles prepared (20), and the nitrocellulose particles used to stimulate
peripheral blood mononuclear cell (PBMCs) in vitro.
A stimulation index (SI) for each molecular weight section was calculated as follows:
SI ⫽ mean CPM experimental wells/mean CPM control wells
Control wells contained nitrocellulose particles without antigen. Positive proliferation was considered to be an SI greater than 2.0, which
corresponds to greater than the mean ⫹ 3 sd of control values (8).
Antigen doses and specificity of PBMC responses of type 1 diabetes
patients to the islet protein preparations and known islet autoantigens
using cellular immunoblotting have been previously described (8).
PBMC responses to tetanus toxoid were used as a control antigen along
with the PBMC responses to mitogens, which were included to test for
viability of the cultures. PBMC responses of type 1 diabetes patients,
autoantibody positive type 2 diabetes patients, autoantibody-negative
type 2 diabetes patients, and controls to tetanus toxoid have been shown
to be similar (11). Based on results in more than 60 controls and 60
patients with type 1 diabetes, cellular immunoblotting is considered
positive when more than three blot sections have an SI greater than
2.0 (12).
HLA typing
DR typing of subjects was performed by the Molecular and Genetics
Core of the Diabetes Endocrinology Research Center at the University
of Washington.
Briefly, DNA was isolated from peripheral blood leukocytes by an
automatic extractor (model 340 A, Applied Biosystems, Foster City, CA)
2224
J Clin Endocrinol Metab, May 2004, 89(5):2222–2227
Brooks-Worrell et al. • Autoreactivity in Children with Diabetes
and the HLA class II typing was performed by an oligoblot hybridization
method (21). DRB1 was amplified by the PCR and labeled by incorporation of digoxigenin-labeled dUTP during the PCR. High-resolution
HLA typing was achieved by the hybridization of the labeled PCR
products to nylon membranes containing allele-specific oligo-probes
selected from various functional DRB loci. Positive reactions were visualized by a chemiluminescent detection method (Roche Diagnostics,
Indianapolis, IN).
Statistics
Autoantibodies and T-cell responses to islet proteins were tested in
a blinded fashion. Clinical classification was determined before knowledge of T-cell or autoantibody status. Unpaired t tests and Fisher’s exact
test were performed to determine significance among groups.
FIG. 1. Summary of autoantibody and T-cell responses for all subjects.
Results
Children clinically classified with type 1 diabetes (n ⫽ 37)
Weight loss was reported in 81% of type 1 subjects, 78%
had urinary ketones, with 19% having acidosis as well. Clinical characteristics are summarized in Table 1.
Thirty-six of the 37 children classified with clinical type 1
diabetes were positive for two or more autoantibodies. One
child was autoantibody negative at diagnosis. Four of the 37
patients (11%) were positive for only two autoantibodies
(three positive for GAD/IA-2 and one positive for ICA/
GAD), 17 of 37 (46%) were positive for only three autoantibodies (11 for ICA/GAD/IA-2 and six for ICA/IAA/IA-2)
and 15 of 37 (41%) were positive for all four autoantibodies
tested (ICA/IAA/GAD/IA-2). Autoantibody results are
summarized in Table 3 and Fig. 1.
Thirty-three of the 37 children (89%) clinically classified as
type 1 diabetes demonstrated T-cell reactivity to islet proteins using cellular immunoblotting (Fig. 2). The one child
that was negative for all four autoantibodies was an Asian
male who demonstrated T-cell reactivity to 13 blot sections.
There was no correlation observed between the magnitude
(SI) of T-cell reactivity and number of autoantibodies
positive.
Children clinically classified with type 2 diabetes (n ⫽ 19)
Eighteen of the 19 type 2 patients were obese (⬎85th percentile) and all 19 patients demonstrated the presence of
acanthosis nigricans. Although none had DKA, 26% of the
TABLE 3. Percentages of patients positive in each group for
autoantibody and T cell responses to islet proteins
Patient
groups
Type 1
(n ⫽ 37)
Type 2
(n ⫽ 19)
ID (n ⫽ 16)
Positive autoantibody
responses (%)
Total Ab
positivity
(%)
Positive T
cell
responses
blots (%)
1
Abs
2
Abs
3
Abs
4
Abs
3
0
11
46
41
97b,c
89d,e
26
21
32
11
11
74b
37d
31
13
19
38
0
68c
38e
0
Absa
ID, Indeterminant patients.
a
Number of autoantibodies positive.
b
Differences between type 1 and type 2 patients, P ⬍ 0.0001.
c
Differences between type 1 and ID patients, P ⬍ 0.001.
d
Differences between type 1 and type 2 patients, P ⬍ 0.005.
e
Differences between type 1 and ID patients, P ⬍ 0.05.
FIG. 2. T-cell responses to islet proteins, using cellular immunoblotting, of children classified with type 1 diabetes, n ⫽ 37; type 2 diabetes, n ⫽ 19; and indeterminant diabetes, n ⫽ 16. Shaded symbols
indicate individuals positive for at least one autoantibody.
patients had urinary ketones, and 32% demonstrated weight
loss (Table 1).
Autoantibody status is summarized in Table 3 and Fig. 1.
Five of the 19 patients (26%) classified with type 2 diabetes
were negative for autoantibodies. Of the 14 patients (74%)
positive for autoantibodies, four patients (21%) were positive
for only one autoantibody [one patient was ICA positive only
(6 JDF units), one patient was IAA only, two patients were
GAD only], six patients were positive for only two autoantibodies (one patient was IAA/GAD, four patients were positive for ICA/IA-2, one patient was ICA/GAD), two patients
were positive for only three autoantibodies (one patient
ICA/GAD/IA-2 and one patient ICA/IAA/IA-2), and two
patients (11%) were positive for all four autoantibodies
tested.
Four of the five patients that were autoantibody negative
were also negative for T-cell responses (Fig. 2). The other
autoantibody-negative patient responded to six blot sections.
This subject was Hispanic, was obese, had acanthosis nigracans, and presented with urinary ketones. Of the 14 patients that were positive for autoantibodies, five were also
positive for T-cell reactivity. Of the five patients that were
positive for T-cell responses, one was positive for GAD autoantibodies only, one for ICA/GAD, one for ICA/GAD/
Brooks-Worrell et al. • Autoreactivity in Children with Diabetes
IA-2, and two were positive for all four autoantibodies. The
other nine patients who were negative for T-cell reactivity,
four patients were positive for ICA/IA-2, one was positive
for ICA/IAA/IA-2, one was positive for ICA (6 JDF units)
only, two were GAD positive only, and one was IAA positive
only.
Children clinically classified with indeterminant
diabetes (n ⫽ 16)
Of the indeterminant patients, 31% were obese, 56% had
urinary ketones, 19% had acanthosis nigracans, 31% had
weight loss, and 19% presented in DKA (Table 1).
Five of the 16 patients (31%) classified with indeterminant
diabetes were negative for autoantibodies (Table 3 and Fig.
1). Of the 11 patients positive for autoantibodies, two were
positive for only one autoantibody (one was positive for ICA,
one for IA-2), three patients were positive for only two autoantibodies (ICA/IA-2), and six were positive for only three
autoantibodies (four for ICA/GAD/IA-2, one for IAA/
GAD/IA-2, and one for ICA/IAA/GAD).
Four of the five autoantibody-negative patients were also
negative for T-cell responses (Fig. 2). The autoantibodynegative patient who was positive for T-cell responses, responding to four blot sections, was a lean Asian with urinary
ketones. Of the 11 patients who were positive for autoantibodies, five were also positive for T-cell responses to islet
proteins. Of these five, one was positive for ICA only, one for
ICA/IAA/GAD, and three were positive for ICA/GAD/
IA-2. None of the three patients that were positive for ICA/
IA-2 demonstrated T-cell reactivity to islet proteins.
Comparisons among type 1, indeterminant, and type 2
diabetes patients
Significant differences were observed between clinically
classified type 1 and type 2 diabetes children for the number
of autoantibodies positive (P ⬍ 0.0001) and the number of
blot sections recognized by their T cells (P ⬍ 0.005). Differences were also observed for the number of autoantibodies
positive (P ⬍ 0.001) between type 1 and indeterminant diabetes patients and for the number of blot sections positive
(P ⬍ 0.05). No significant differences were observed between
type 2 and indeterminant patients in autoantibody or T-cell
reactivity. Autoantibody and T-cell reactivity to islet proteins
are summarized in Table 3 for the three groups of patients.
DR typing was available on 28 type 1 patients, 15 type 2
patients, and 11 indeterminant patients. Eighty-nine percent
(25 of 28) of the type 1 patients had diabetes-associated HLA
(DR3 and/or DR4). Of these 25, 24 were autoantibody positive. Of the type 2 patients, 67% (10 of 15) were positive for
DR3 and/or DR4 and eight of the 10 were autoantibody
positive. For the indeterminant patients, 82% (9 of 11) were
found to have DR3 and/or DR4, and six of these nine were
autoantibody positive. The frequency of DR3 and/or DR4 in
the three groups was not significantly different, but this may
be due to the relatively small number of subjects in each
group. The DR typing and autoantibody data are summarized in Table 4.
We also questioned whether clinical phenotypic characteristics such as obesity, urinary ketones, DKA, acanthosis
J Clin Endocrinol Metab, May 2004, 89(5):2222–2227
2225
TABLE 4. Number of available patients with diabetes-associated
HLA (DR3 and/or DR4) and autoantibody status
Type of subject
Autoantibody
status
DR3 and/or DR4
Type 1
(n ⫽ 28/37)a
Type 2
(n ⫽ 15/19)a
Indeterminant
(n ⫽ 11/16)a
Ab(⫹)
Ab(⫺)
Ab(⫹)
Ab(⫺)
Ab(⫹)
Ab(⫺)
24
1
8
2
6
3
a
Total number of subjects in each patient group available for HLA
testing out of the total for each group.
TABLE 5. Clinical characteristics vs. autoantibody responses and
T cells responses
Characteristic
Weight loss
Obesity
Urinary ketones
DKA
Acanthosis nigricans
Ethnicity
Diagnosis
Type 1
Indeterminant
Type 2
Positive T cells or
positive Ab (n ⫽ 64)
Negative T cells and
negative Ab (n ⫽ 8)
38
22
41
9
16
22
3
5
1
1
6
4
37
12
15
0
4
4
nigricans, or ethnicity would help segregate the nonautoimmune diabetic children from those presenting with islet autoimmunity. When we divided the subjects into those positive for T-cell responses or positive autoantibody responses
(n ⫽ 64) vs. those without T cells and autoantibody positivity
(n ⫽ 8), we observed that none of the clinical characteristics
[with the exception of the presence of urinary ketones (P ⬍
0.05)] were able to identify children with vs. without autoimmunity. These results are shown in Table 5.
Discussion
The diagnosis of clinical diabetes in children over the years
has been overwhelmingly a diagnosis of type 1 diabetes.
However, in recent years an increase in children diagnosed
with type 2 or indeterminant diabetes (admixture of characteristics) has been reported (1– 4). Most recently, concerns
have been raised that some of the children diagnosed with
type 2 diabetes may actually be misdiagnosed based on clinical features (obesity, acanthosis nigracans) and actually represent obese children with autoimmune diabetes (5, 6, 22–24).
Thus, in our study, we investigated how commonly children
clinically classified with type 2 or indeterminant diabetes had
autoantibody reactivity to islet proteins, compared with children with classical type 1 diabetes. We also evaluated these
patients for T-cell reactivity to islet proteins commonly observed in children with clinically classified type 1 diabetes (6,
8, 11).
In this study, we observed that all but one of the children
classified with classic type 1 diabetes (n ⫽ 37) were positive
for autoantibodies to islet proteins, whereas 74% of the type
2 and 69% of the indeterminant patients were positive for
islet cell autoantibodies. In our previous studies of adults
with phenotypic type 2 diabetes (11, 25), we observed a much
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J Clin Endocrinol Metab, May 2004, 89(5):2222–2227
lower frequency of autoantibodies to islet proteins. Thus, the
high percentage of children classified with type 2 or indeterminant diabetes who were positive for autoantibodies was
an unexpected result. However, based on the sensitivity and
specificity of the autoantibody assays we employed (see Subjects and Methods), we are confident in the results we observed. Because positivity for autoantibodies has been set at
the 99th percentile of normal controls, we would expect only
1% of the apparent autoantibodies observed to be false positives. We also investigated whether the presence of autoimmunity was evident in these patients by investigating
T-cell reactivity and HLA of the patients. The T-cell assay that
we used, cellular immunoblotting, recently performed well
at distinguishing controls from recently diagnosed type 1
diabetes patients in a blinded study funded by National
Institutes of Health (data not published).
A high percentage of type 2 and indeterminant patients
demonstrated autoimmunity as determined by T-cell reactivity to islet proteins. The diabetes susceptibility alleles HLA
DR3 and/or DR4 exist in approximately 44% of the general
population. In this study, we found these susceptibility alleles in 89% (25 of 28) of type 1 patients, 67% (10 of 15) of type
2 patients, and 82% (9 of 11) of indeterminant patients available for typing. This high percentage of subjects with diabetes-associated HLA in the type 2 and indeterminant diabetes groups further supports the autoantibody and T-cell
results that a large number of patients identified by physicians as type 2 diabetes in fact demonstrate autoimmunity.
Therefore, taken together the autoantibody, T-cell, and HLA
status of these patients support the conclusion that many
patients classified as either type 2 or indeterminant may have
autoimmune diabetes. Clinical characteristics did not clearly
distinguish type 1 vs. type 2 or indeterminant diabetes.
Our results report a higher percentage of patients classified with type 2 or indeterminant diabetes demonstrating
autoreactivity to islet proteins than those reported by others
(5, 6, 26); however, this appears to most likely be due to
different patient populations studied. In our study 26% of
our type 2 patients were Caucasian, 21% Hispanic, 11%
Asian, 11% African-American, and 5% other. Whereas patients in the studies by Libman et al. (6) and Katz et al. (26)
were primarily African-American (67 and 51.7%, respectively), and in a study by Hathout et al. (5), the type 2 patient
population studied was 62.9% Hispanic. The percentage of
patients with autoimmunity appears to be strongly influenced by race; thus, varying patient populations may significantly vary the percentages of patients positive for autoreactivity. However, the important issue is the relatively
high percentage of patients diagnosed with type 2 or indeterminant diabetes that are positive for islet cell autoimmunity in all these studies.
Another interesting observation concerning autoantibodies involved a cohort of seven children, three diagnosed with
indeterminant diabetes and four with type 2 diabetes. All
seven of these children were positive for ICA/IA-2 but did
not demonstrate T-cell reactivity to islet proteins. Five of the
seven children were obese and demonstrated the presence of
acanthosis nigricans. Future studies may help us identify
whether children with ICA and IA-2 alone, or possibly the
absence of GAD autoantibodies in children, represent a sub-
Brooks-Worrell et al. • Autoreactivity in Children with Diabetes
group of patients with potentially unique autoimmune characteristics. Seissler et al. (27) observed that the presence of
IA-2 autoantibodies, restricted to the IgG4 subclass, may in
fact be associated with protection from type 1 diabetes. Further studies will help to confirm whether the patient population in our study has a similar antibody restriction.
In this study, we also observed T-cell responses to islet
proteins in children with type 2 and indeterminant diabetes.
Taken together, the results of our study and others demonstrate that autoreactivity to islet proteins, whether autoantibodies or T cells or both, is more prevalent than expected
in children classified with type 2 or indeterminant diabetes.
Moreover, these studies also demonstrate that classic autoantibody-negative type 2 diabetes may be less common than
believed in patients with diabetes diagnosed during childhood. We also found that diabetes-associated HLA (DR3
and/or DR4) occurred more commonly in the autoantibodypositive patients in each of the three groups. The presence of
diabetes-associated HLA, however, is also present in some of
the autoantibody-negative patients. Thus, having diabetesassociated DR alleles is not a strong marker of type 1 diabetes
or of autoimmunity.
Katz et al. (26) observed that children with new-onset type
2 diabetes could be distinguished from those with type 1
diabetes with a combination of age, race, body mass index,
acanthosis nigracans, c-peptide, and urine ketones. Hardin et
al. (28) observed that type 2 diabetes in youth could be
distinguished from type 1 diabetes by age, ethnicity, obesity,
presence of acanthosis nigricans, serum pH, Tanner stage,
and family history. In our study we compared obesity, presence of acanthosis nigricans, ethnicity, weight loss, diabetic
ketoacidosis, and urine ketones. We observed that none of
the clinical characteristics, with the possible exception of
urine ketones, segregated those patients with islet autoimmunity from those patients who did not have islet autoimmunity. The presence of ketosis (as did Katz et al.) significantly correlated (P ⬍ 0.05) with the presence of autoimmunity to islet proteins, but the absence of ketones did not
indicate the absence of autoimmunity to islet proteins. Two
of the nonobese patients (one type 2 and one indeterminant)
demonstrated acanthosis nigricans. Both of these patients
were positive for autoantibody, T cells, urinary ketones, and
weight loss. One of the two patients presented in DKA. Thus,
it would be of interest to investigate what level of insulin
resistance may be present in these two patients.
In adults, most patients with phenotypic type 2 diabetes
are autoantibody and T cell negative. However, this does not
appear to be the case in children. Our unexpected results
demonstrate that autoimmunity to islet proteins consistent
with type 1 diabetes is present in a high percentage of children clinically classified as type 2 or indeterminant diabetes.
In agreement with the adult autoantibody-positive type 2
patients, clinical phenotypic characteristics typically associated with a diagnosis of type 2 diabetes do not reliably
distinguish autoimmune vs. nonautoimmune children (25).
These results suggest that the reported increase in clinical
type 2 diabetes may actually represent an increase in obese
children with type 1 diabetes and that classic type 2 diabetes
(negative for autoantibodies and T-cell autoreactivity to islet
proteins) is less common. We are currently investigating
Brooks-Worrell et al. • Autoreactivity in Children with Diabetes
whether the clinical course is different in children who are
autoantibody and T cell positive or negative for both or cases
in which autoantibodies and T-cell results are different. Such
a study requires a much larger number of patients followed
up prospectively and is currently underway. Currently, there
is a 5-yr multicenter study (SEARCH) funded by the Centers
for Disease Control and Prevention and the National Institute of Diabetes and Digestive and Kidney Diseases investigating how types 1 and 2 diabetes differ in children by age
and race/ethnicity. With more studies in the future incorporating autoantibody and T-cell assays to study diabetes in
children, the questions concerning the presence of autoreactivity in these patients can be answered.
One of the extreme difficulties, as demonstrated by the
results in this study, is the identification of autoantibodynegative type 2 children. Thus, a much larger study will be
needed to identify a large number of autoantibody-negative
subjects. The results of this current study stress the need for
more studies to investigate the presence vs. the absence of
autoimmune markers in children with diabetes, even if the
clinical features strongly suggest a diagnosis of type 2 diabetes. Our results also demonstrate that children with diabetes should be tested for the presence of HLA, autoantibodies, and T cells reactive with islet proteins, regardless of
their clinical presentation. Those children with islet autoimmunity should probably not be classified as type 2 diabetes
patients.
Acknowledgments
We sincerely thank Rick Mauseth, M.D., for providing some of the
diabetes patients. Also, we thank Connaught Laboratories for supplying
the tetanus toxoid. We also thank Dr. G. Eisenbarth (Barbara Davis
Research Center, Denver, CO) for supplying the plasmid containing the
cDNA coding for the cytoplasmic portion of IA-2.
Received August 4, 2003. Accepted February 4, 2004.
Address all correspondence and requests for reprints to: Barbara
Brooks-Worrell, Ph.D., 1660 South Columbian Way (111), DVA Puget
Sound Health Care System, Seattle, Washington 98108. E-mail:
[email protected].
This work was supported in part by the Medical Research Service of
the Department of Veterans Affairs through a VA Merit Review grant
and by the following grants from the NIH: MO1 RR00037-41, P01
DK53004, and P30 DK17047.
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