The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 9 3348-3351
Copyright © 2000 by The Endocrine Society
Tumor Necrosis Factor Microsatellite Polymorphism Influences the Development of Insulin Dependency in Adult-Onset Diabetes Patients with the DRB1*1502-DQB1*0601 Allele and Anti-Glutamic Acid Decarboxylase Antibodies
Hiroshi Obayashi,
Goji Hasegawa,
Michiaki Fukui,
Kenji Kamiuchi,
Akane Kitamura,
Masakazu Ogata,
Toshihiro Kanaitsuka,
Hirofumi Shigeta,
Yoshihiro Kitagawa,
Koji Nakano,
Masataka Nishimura,
Mitsuhiro Ohta and
Naoto Nakamura
From The First Department of Internal Medicine, Kyoto Prefectural
University of Medicine (H.O., G.H., M.F., K.K., A.K., M.O., H.S., T.K.,
Y.K., K.N., N.N.), Kyoto 602-0841; Kyoto Microbiological Institute
(H.O.), Kyoto; Department of Clinical Research Center, Utano National
Hospital (M.N., M.O.), Kyoto; and Department of Clinical Chemistry,
Kobe Pharmaceutical University (M.O.), Kobe, Japan
Address correspondence and requests for reprints to: Dr. Goji Hasegawa, The First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan. E-mail: goji{at}koto.kpu-m.ac.jp
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Abstract
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Recently, several studies have demonstrated that tumor necrosis factor
microsatellite polymorphism (TNFa) contributes to the
susceptibility of type 1 diabetes. This study investigates the
influence of TNFa on the predisposition to insulin
dependency in adult-onset diabetic patients with type 1
diabetes-protective human leukocyte antigen haplotypes. The
TNFa of three groups of DRB1*1502-DQB1*0601-positive
diabetic patients who had initially been nonketotic and noninsulin
dependent for more than 1 yr was analyzed. Group A included 11
antibodies to glutamic acid decarboxylase (GADab)-positive patients who
developed insulin dependency within 4 yr of diabetes onset. Group B
included 11 GADab-positive patients who remained noninsulin dependent
for more than 12 yr. Group C included 12 GADab-negative type 2
diabetes, and a control group included 18 nondiabetic subjects. In the
group C and control subjects, DRB1*1502-DQB1*0601 was strongly
associated with the TNFa13 allele. DRB1*1502-DQB1*0601
was strongly associated with the TNFa12 allele among the
group A patients, but not among the group B patients. Interestingly,
sera from all patients with non-TNFa12 and
non-TNFa13 in group B reacted with GAD65 protein by Western
blot. These results suggest that TNFa is associated with a
predisposition to progression to insulin dependency in
GADab/DRB1*1502-DQB1*0601-positive diabetic patients initially
diagnosed with type 2 diabetes and that determination of these
patients’ TNFa genotype may allow for better prediction of
their clinical course.
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Introduction
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TYPE 1 (INSULIN-DEPENDENT) diabetes
mellitus is considered to be an organ-specific autoimmune disease, and
both genetic and environmental factors contribute to the development of
this disease. Autoantibodies to glutamic acid decarboxylase (GADab)
(1) have both diagnostic and predictive value as
immunological markers for type 1 diabetes, not only in juvenile-onset
type 1 diabetes but also in adult-onset diabetic patients initially
diagnosed with type 2 diabetes (2, 3, 4, 5, 6). However, GADab also
occurs in typical type 2 diabetes patients, thus, not all
GADab-positive diabetic patients develop insulin dependency. Although
different gene alleles and haplotypes are associated with type 1
diabetes in different ethnic groups, certain HLA-DR and -DQ haplotypes
are associated with the susceptibility to type 1 diabetes. The HLA
DRB1*1501-DQB1*0602 haplotype confers strong protection from
insulin-dependent diabetes mellitus in Caucasians. Pugliese et
al. (7) have reported that DQB1*0602 is associated
with dominant protection from diabetes even among islet cell
antibody-positive first-degree relatives of patients with type 1
diabetes. Among Japanese, the haplotypes HLA DRB1*1502-DQB1*0601
are known to confer protection from type 1 diabetes. However, the age
of onset for type 1 diabetes is variable, and it has been reported that
the frequencies of some type 1 diabetes susceptibility HLA class II
alleles and/or haplotypes vary depending on the age of disease onset
(8, 9). Thus, the protection conferred by DQB1*0602
and/or *0601 decreases with increasing age of onset of disease.
Tumor necrosis factor (TNF) gene is located in the central region of
the major histocompatibility complex, and some polymorphic
microsatellites have been identified in and near the TNF gene
(10). It has been reported that the TNF microsatellite
(TNFa) polymorphism might be involved in susceptibility to
type 1 diabetes (11, 12, 13). Recently, we have reported that
the TNFa polymorphism is associated with age-at-onset of
type 1 diabetes (14).
In the present study, we investigated the influence of TNFa
on the predisposition to insulin dependency in
GADab/DRB1*1502-DQB1*0601-positive adult-onset (>35 yr of age)
diabetic patients who had initially been nonketotic and noninsulin
dependent for more than 1 yr. We also investigated the reactivity of
GADab by Western blot analysis and the prevalence of a mitochondrial
DNA mutation at bp 3243 (mtDNA A3243G gene mutation).
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Subjects and Methods
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Subjects
GADab/DQB1*1502-DQB1*0601 positive initially diagnosed with
type 2 (noninsulin-dependent) diabetes patients who developed insulin
dependency (group A: n = 11; six men and five women; mean age,
56 ± 3 yr; mean age at onset; 44 ± 3 yr) and an additional
group that remained noninsulin dependent (group B: n = 11; five
men and six women; mean age, 57 ± 3 yr; mean age at onset;
45 ± 2 yr) were identified through GADab screening of our study
of approximately 4000 type 2 diabetic patients, as partially previously
reported (6). Twelve GADab-negative type 2 diabetic
patients with DQB1*1502-DQB1*0601 (group C: 6 men and 6 women; mean
age, 57 ± 5 yr; mean age at onset; 44 ± 6 yr) and 18
GADab-negative nondiabetic subjects with DQB1*1502-DQB1*0601 (10
men and 8 women; mean age; 44 ± 7 yr) selected from the same
study served as controls. All the subjects were Japanese and unrelated
and residents of the Kyoto and Osaka area. The decision to start
insulin therapy was made by the patient’s physician in accordance with
routine clinical guidelines. These included a fasting blood glucose
level of more than 12.2 mmol/L and/or HbA1c more than 8.5% in the
presence of the maximum daily dose of gliebenclamide (10 mg), and
glucagon-stimulated C-peptide values less than 0.6 nM (6
min after iv injection of 1 mg glucagon).
HLA-DRB1 and -DQB1 typing anti-GAD antibody assay
HLA-DRB1 and -DQB1 alleles were determined using a PCR-low ionic
strength single-stranded conformation polymorphism method as described
by Maruya et al. (15) and/or the
PCR-sequence-specific oligonucleotide method according to the 11th
International Histocompatibility Workshop protocol
(16).
Anti-GAD antibody assay
Anti-GAD antibody was assayed using a radioimmunoprecipitation
assay kit using 125I-labeled human recombinant
GAD65 (Cosmic Co. Ltd., Tokyo, Japan) as previously
described (17). This anti-GAD antibody assay was entered
into the international Antibody Proficiency Program [’96 Workshop,
organized by J. Bertulfo (University of Florida, Gainesville, FL)].
The assay achieved a specificity of 100% and a sensitivity of
100%.
mtDNA A3243G gene mutation analysis
Genomic DNA was extracted from peripheral blood with a DNA
extractor WB kit (Wako pure chemical Ltd., Osaka, Japan) according to
the manufacturer’s instructions. Analysis of mtDNA A3243G mutation was
performed by PCR-restriction fragment length polymorphism method as
described previously (18).
Analysis of TNFa microsatellite polymorphism
Polymorphism of TNFa microsatellite alleles was analyzed as
previously reported (14). Briefly, TNFa microsatellite was
amplified in the PCR using the primers described by Udalova
et al. (10): forward
(5'-GCCTCTAGATTTCATCCAGCCACA-3') and reverse (5'-CCTCTCTCCCCTGCAACAC
ACA-3'). The forward primer was 5' end-labeled with a fluorescent dye
(6-Fam). The amplification was performed in a 50-µL reaction with 50
ng genomic DNA and 1.0 U AmpliTaq Gold polymerase (Perkin-Elmer Corp.,
Norwalk, CT). The initial denaturation step was 10 min, followed by 35
cycles of 94 C for 1 min, 60 C for 50 s, and 72 C for 1 min. The
PCR products were loaded onto a 6% denaturing polyacrylamide gel with
an internal lane standard labeled with Genescan-500 Rox dye and
analyzed on an ABI 373 DNA sequencing system using the Genescan 672
software (Applied Biosystems, Foster City, CA).
Analysis of GADab by Western blot
The analysis of GADab by Western blot was performed using
recombinant GAD65. Briefly, purified recombinant
GAD65 protein [a kind gift from Dr. M. Powell (RSR Ltd.,
Cardiff, UK)] (19) was separated by electrophoresis on a
4–20% gradient SDS-polyacrylamide slab gel according to the procedure
of Laemmli (20) and electrophoretically transferred to a
nitrocellulose membrane according to the procedure of Towbin et
al. (21). After blocking, the membrane was cut into
strips and incubated for 2 h in a 1:100 dilution of serum from
patients at 20 C. After washing with phosphate-buffered saline (pH 7.4)
containing 0.02% Tween 20, strips were incubated for 2 h in
horseradish peroxidase-conjugated antihuman IgG (1:2000 dilution;
Bio-Rad Laboratories, Richmond, CA) at 20 C and washed with washing
buffer. The reaction products were visualized using the substrate
4-chloro-1-naphthol.
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Results
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Table 1
summarizes the primary
results of the study. In the group C and non-diabetic subjects who
served as controls, the haplotype DRB1*1502-DQB1*0601 was
strongly associated with the TNFa13 allele, suggesting
a linkage disequilibrium between them. In contrast, the haplotype
DRB1*1502-DQB1*0601 was strongly associated with the
TNFa12 allele in group A (10 of 11 patients possessed
TNFa12 allele). None of the group C patients possessed
TNFa12 (two patients possessed TNFa13 and nine
patients possessed non-TNFa12 and non-TNFa13
alleles).
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Table 1. TNFa polymorphisms in HLA
DRB1*1502/DQB1*0601-positive diabetic patients and nondiabetic
control subjects
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All sera (n = 9) from patients with non-TNFa12 and
non-TNFa13 alleles in group B reacted with GAD65
protein on Western blots. In contrast, only 2 of 13 (15%) sera from
diabetic patients with TNFa12 or TNFa13 were
GADabpositive by Western blots.
None of the patients had a mtDNA A323G gene mutation.
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Discussion
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To our knowledge, the present study is the first attempt to
examine the polymorphic microsatellites of the TNF gene in
GADab-positive diabetic patients with type 1 diabetes-protective HLA
DRB1-DQB1 genotypes. A major finding of this study is that the
distribution of TNFa alleles was different between
GADab-positive diabetic patients and GADab-negative subjects, including
both diabetic patients and healthy controls. This result suggests that
TNFa polymorphism is associated with the production of
GADab. More noteworthy is that the distribution of the TNFa
allele was markedly different between GADab-positive patients who
developed insulin-dependent and GADab-positive patients who remained
noninsulin dependent. Ten of the 11 (91%) GADab-positive patients who
developed insulin dependency had the TNFa12 allele, whereas
none of the 11 GADab-positive patients who did not develop insulin
dependency had the TNFa12 allele. This finding indicates
that the TNFa12 allele is associated with the predisposition
to insulin dependency in GADab-positive patients with
DRB1*1502-DQB1*0601 who are initially diagnosed with type 2
diabetes. Recently, Pociot et al. (11) and we
(14) have shown that TNFa secretion capacity is related to
TNFa polymorphism. The actions of TNFa include neutrophil
activation, increased T and B cell proliferation, immunoglobulin
synthesis, up-regulation of HLA class I and II expression, and
induction of adhesion molecules (22). Interestingly, our
Western blot data suggests a significant association between
TNFa polymorphism and the heterogeneity of GADab. It has
been reported that most sera from type 1 diabetic patients recognize
only conformational epitopes in the GAD65 molecule and that
sera able to react with denatured GAD protein by Western blot are rare
from typical type 1 diabetic patients (1).
Concerning the development of insulin-dependency in adult-onset
diabetes, Oka et al. (23) and Kobayashi
et al. (24) have reported that the mtDNA A3243G
gene mutation was associated with the development of insulin dependency
in adult-onset diabetic patients with islet cell antibodies. However,
none of the patients has the mtDNA A3243G mutation in our present
study.
In conclusion, the results in this study indicate that TNFa
polymorphism is associated with the predisposition to insulin
dependency in GADab/DRB1*1502-DQB1*0601-positive diabetic patients
initially diagnosed with type 2 diabetes. This suggests that analysis
of TNFa polymorphism may be useful for predicting the
clinical course of GADab/DRB1*1502-DQB1*0601-positive diabetic
patients initially diagnosed with type 2 diabetes, allowing
better-informed decisions for treatment.
Received May 2, 2000.
Accepted June 14, 2000.
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Abstract
Introduction
