The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 653-655
Copyright © 2001 by The Endocrine Society
The Codon 17 Polymorphism of the CTLA4 Gene in Type 2 Diabetes Mellitus1
Harald Rau,
Jens Braun,
Horst Donner,
Jochen Seissler,
Thorsten Siegmund,
Klaus H. Usadel and
Klaus Badenhoop
Medical Department I, Centre of Internal Medicine, Klinikum of the
Johann Wolfgang Goethe-University, 60590 Frankfurt/Main, Germany
Address correspondence and requests for reprints to: Prof. Dr. med. Klaus Badenhoop, Medizinische Klinik I, Zentrum der Inneren Medizin, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. E-mail:
badenhoop{at}em.uni-frankfurt.de
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Abstract
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Several studies have demonstrated an association of CTLA4 (IDDM12)
alanine-17 with type 1 diabetes, but CTLA4 variants have not yet been
investigated in type 2 diabetes. The CTLA4 exon 1 polymorphism (49 A/G)
was analyzed in 300 Caucasian patients with type 2 diabetes and 466
healthy controls. All patients were negative for glutamate
decarboxylase and islet cell antibodies. CTLA4 alleles were
defined by PCR, single-strand conformational polymorphism, and
restriction length fragment polymorphism analysis using
BbvI. The distribution of alleles as well as the
genotypic and phenotypic frequencies were similar among patients and
controls [AA, 42 vs. 39%; AG, 47 vs.
46%; GG, 11 vs. 15%, P = not
significant (n.s.); A/G, 65/35% vs. 62/38%,
P = n.s.; alanine/threonine 92/58%
vs. 85/61%, P = n.s.]. However,
detailed analysis of clinical and biochemical parameters revealed a
tendency of GG (alanine/alanine) toward younger age at disease
manifestation (46.8 ± 0.8 vs. 49.5 ± 0.8 yr,
mean ± SEM), lower body mass index (21.4 ± 0.5
vs. 24.4 ± 0.5 kg/m2,
P = 0.042), and basal C-peptide level (0.33 ±
0.07 vs. 0.53 ± 0.07nmol/L), as well as earlier
start of insulin treatment (5.8 ± 1.2 vs. 8.7
± 0.6 yr) and higher portion of patients on insulin (71
vs. 61%). Patients with the AA genotype were
significantly less likely to develop microangiopathic lesions
(P < 0.0005). No differences were found for
hypertension or family history of type 2 diabetes. In conclusion, CTLA4
alanine-17 does not represent a major risk factor for type 2 diabetes.
Additional studies on larger groups and different ethnic groups are
warranted to clarify the association of the GG genotype with faster
ß-cell failure and the lower rate of microvascular complications in
AA carriers.
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Introduction
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TYPE 2 DIABETES consists of specific and
definable subtypes, and a reasonable number of underlying gene defects
has been identified by the examination of candidate genes. Whereas
several studies have demonstrated an association of CTLA4 (IDDM12)
alanine-17 with type 1 diabetes (1, 2, 3, 4, 5, 6), CTLA4 variants
have not yet been investigated in type 2 diabetes. CTLA4 has been
mapped on chromsome 2q33 and named IDDM12 (1, 7). This
region contains the CTLA4 (cytotoxic T lymphocyte antigen 4) gene that
encodes a receptor on T cells interacting with the B7 accessory
molecules. CTLA4 represents a key regulatory element in the T
cell/antigen-presenting cell interaction (reviewed in Ref.
8). Because CTLA4 mediates antigen-specific apoptosis
(9) and progressive ß-cell failure is a typical feature
of type 2 diabetes, CTLA4 may be a candidate gene to confer
susceptibility also to type 2 diabetes.
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Subjects and Methods
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Subjects
The codon 17 polymorphism in the CTLA4 gene was studied in 300
patients with type 2 diabetes and 466 nondiabetic controls. All
patients were Caucasians and negatively tested for glutamate
decarboxylase, insulin, and islet cell antibodies. IA-2
antibodies were not studied because all patients were older than 36 yr
at time of diagnosis. Healthy controls were randomly collected among
Caucasian blood donors and had no family history for diabetes. The
group of controls included individuals from our previous studies
(2, 10). Extensive clinical and biochemical data on the
patients were obtained with respect to gender, age, age at diagnosis,
relatives with diabetes, antidiabetic treatment, time between disease
manifestation and initiation of insulin treatment, body mass index,
HbA1c, basal C-peptide, diabetic complications,
and blood pressure.
Methods
The CTLA4 exon 1 position 49 (codon 17) polymorphism was defined
as described previously (2). Briefly, PCR was performed
with oligonucleotides forward 5'-GCTCTACTTCCTGAAGACCT-3' and reverse
5'-AGTCTCACTCACCTTTGCAG-3', designed according to the published human
CTLA4-cDNA sequence (11), using 0.2 µg genomic DNA, 1 U
Taq Polymerase (Life Technologies, Inc., Karlsruhe, Germany), 20 pmol of each primer, and 8 mmol dNTPs (4 min
at 94 C, then 30 cycles with 45 sec at 58 C, 45 sec at 72 C, 45 sec at
94 C, and, finally, 4 min at 72 C).
Single-strand conformational polymorphism analysis of CTLA4
polymorphisms
PCR products were screened for variants by Single-strand
conformational polymorphism. Aliquots of the PCR product (2 µL) were
mixed with 2.3 µL deionized formamide, incubated for 5 min at 95 C,
and loaded onto an 8% polyacrylamide gel. Gel electrophoresis was
carried out at 10 mA (10 watts; maximum, 1000 V) for 2.5 h,
keeping constantly 8 C on a Multiphor II apparatus and a Multitemp
cooling system (LKB Pharmacia, Freiburg, Germany). Gels
were silver-stained to vizualize variant conformational fragments,
which correspond to nucleotide substitutions as confirmed by
restriction enzyme analysis, where the restriction enzyme
BbvI cutted the amplificate in the presence of a G at
position 49 (88/74-bp fragments). For restriction length fragment
polymorphism, DNA fragments were resolved in 2.0% agarose gels stained
with SYBR Green I (Molecular Probes, Inc., Leiden,
Netherlands).
Definition of HLA DQA1 and DQB1 alleles
Patients were typed for HLA DQA1 and DQB1 alleles as described
previously (12).
Statistical analysis
The Mann-Whitney U test, the
2 test with Yates’ correction, and Fisher’s
exact test were used for statistical analysis where appropriate.
P values were multiplied by the numbers of tests
(pcorr). Statistical significance was defined at
P less than 0.05.
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Results
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The CTLA4 exon 1 polymorphism was defined in all 300 patients and
466 controls. The distribution of alleles as well as the genotypic and
phenotypic frequencies were similar among patients and controls (Table 1
). However, detailed analysis of
clinical and biochemical parameters revealed a tendency of GG
(alanine/alanine) carriers toward younger age at disease manifestation,
lower body mass index, and basal C-peptide level as well as earlier
start of insulin treatment and higher portion of patients on insulin.
The difference in body mass index reached statistical significance (GG
vs. AA/AG, 21.4 vs. 24.4
kg/m2, pcorr = 0.042). No
differences were found for family history of type 2 diabetes and
hypertension (Table 2 and data not shown,
respectively). The investigation of the HLA DQA1 and DQB1 alleles,
which confer strong susceptibility to type 1 diabetes
(12), demonstrated a similar distribution of HLA alleles
and haplotypes among carriers of the AA, AG, and GG genotypes (Table 3 and data not shown). However, GG
carriers were significantly more often positive for HLA DQB110602
(pcorr = 0.04), which is considered to confer
protection against type 1 diabetes mellitus (12) (Table 3). Renal and multiple microangiopathic diabetic complications were
significantly less frequent among AA carriers (nephropathy: AA
vs. AG/GG, 22 vs. 38%,
pcorr = 0.02; multiple microangiopathic lesions:
AA vs. AG/GG, 9 vs. 29%, pcorr <
0.0005; Table 4). C-peptide levels
correlated inversely with the number of diabetic complications:
C-peptide levels were 0.56 ± 0.60 nmol/L in 124 (42%) patients
without any diabetic complications, 0.50 ± 0.46 nmol/L in 110
(37%) patients with one diabetic lesion, and 0.43 ± 0.56 nmol/L
in 60 (20%) patients with multiple diabetic complications (mean
± SD, P = not significant).
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Table 3. HLA DQA1 and DQB1 high risk alleles as well as
DQA1/DQB1 high risk haplotypes in patients with type 2 diabetes
according to their CTLA4 genotype (243 of 266 patients with the AA or
AG genotype and 33 of 34 patients with the GG genetype could be
analysed for HLA DQA1 and DQB1 alleles as well as for DQA1/DQB1
haplotypes)
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Discussion
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There is increasing evidence that CTLA4 gene polymorphisms confer
susceptibility to several endocrine autoimmune disorders
(e.g. IDDM, Graves’ disease, Hashimoto’s thyroiditis, and
Addison’s disease), as shown by others and ourselves (1, 2, 10, 13). The investigated patients were all negative for glutamate
decarboxylase, insulin, and islet cell antibodies. HLA DQ high-risk
alleles for type 1 diabetes were found with similar frequencies in the
patients of the present study as in our previously published controls
(12). This applied to the whole patient group as well as
to the subgroup of GG-positive patients. Thereby, the protective allele
HLA DQB110602 was even more frequent in GG-positive patients compared
with AA or AG carriers (pcorr = 0.04). Data on
residual insulin secretion was available for 32 of the 34 patients with
the GG genotype, and preserved insulin secretion could be detected in
all of these 32 patients. Hence, only patients with type 2 diabetes
were included in the present study. There was no correlation of CTLA4
alanine-17 with type 2 diabetes in these individuals. However, the GG
genotype was associated with younger age at disease manifestation,
lower body mass index, and basal C-peptide level as well as earlier
start of insulin treatment and higher portion of patients on insulin
without any correlation to HLA DQA1/DQB1 high-risk alleles. In
contrast, the AA genotype was associated with higher basal C-peptide
levels and a lower frequency of microvascular complications. In
conclusion, CTLA4 alanine-17 does not represent a major risk factor for
type 2 diabetes. However, additional studies on larger groups and
different ethnic groups are warranted to clarify the association of the
GG genotype with faster ß-cell failure as well as the lower frequency
of microangiopathic lesions in patients carrying AA. Extending these
findings from the observations made in type 1 diabetes, CTLA4 or a
closely mapping gene may be linked to defective insulin secretion also
in a subgroup of patients with type 2 diabetes.
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Footnotes
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1 Supported by the Deutsche Forschungsgemeinschaft (DFG Ba
976/8-1). 
Received August 16, 2000.
Revised October 18, 2000.
Accepted October 25, 2000.
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References
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Abstract
Introduction
