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Original Studies |
Gene in Anti-Islet Autoantibody-Negative Japanese Patients with Type 1 Diabetes
First Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki 852-8501; and Department of Pediatrics, Kitasato University School of Medicine (N.O., N.M.), Sagamihara 228-8555, Japan
Address all correspondence and requests for reprints to: Eiji Kawasaki, M.D., First Department of Internal Medicine, Nagasaki University School of Medicine, 1–7-1 Sakamoto, Nagasaki 852-8501, Japan. E-mail: f1196{at}cc.nagasaki-u.ac.jp
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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(HNF-1) gene are the
cause of maturity-onset diabetes of the young type 3 (MODY 3), which is
characterized by a severe impairment of insulin secretion and early
onset of the disease. Although the majority of patients with type 1
diabetes have type 1A, immune-mediated diabetes, there is a significant
percentage of the patients who have no evidence of an autoimmune
disorder at the onset of disease. The aim of this study was to estimate
the prevalence of MODY 3 in antiislet autoantibody negative patients
with type 1 diabetes. From a large population-based sample of unrelated
Japanese patients with type 1 diabetes, 28 patients who lacked
autoantibodies to glutamic acid decarboxylase, islet cell antigen
512/insulinoma-associated antigen-2, phogrin (phosphate homolog of
granules of insulinoma)/insulinoma-associated antigen-2ß, and insulin
at the onset of type 1 diabetes were examined by PCR-based direct
sequencing of the 10 exons, flanking introns, and the promoter region
of the HNF-1 gene. Two (7.1%) of 28 autoantibody-negative patients
with type 1 diabetes were identified as carrying mutations in the
HNF-1 gene. One patient carried a frameshift mutation
(Pro379fsdelCT) in exon 6, and another patient carried a
novel 2-bp substitution at nucleotides +45 (G to A) and +46 (C to A)
from the transcriptional site of the promoter region. These mutations
were identified in heterozygous form and were not identified in 64
unrelated healthy control subjects or 54 unrelated islet
autoantibody-positive patients with type 1 diabetes. Functional
analysis of the mutant HNF-1 gene indicated that the
Pro379fsdelCT mutation had no transcriptional
trans-activation activity and acted in a dominant
negative manner. The +45/46 GC to AA mutation in the promoter region
showed reduced promoter activity by 10–20% compared to the wild-type
sequence. In conclusion, about 7% of Japanese diabetic patients
lacking antiislet autoantibodies initially classified as having type 1
diabetes could have diabetes caused by mutations in the HNF-1 gene. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Maturity-onset diabetes of the young (MODY) is a monogenic form of
diabetes characterized by autosomal dominant inheritance, an early age
of onset (usually <25 yr of age), and ß-cell dysfunction (4, 5).
MODY is genetically heterogeneous, resulting from mutations in at least
five genes, the hepatocyte nuclear factor-4 (HNF-4) for MODY1
(6), glucokinase for MODY2 (7), HNF-1 for MODY3 (8), the insulin
promoter factor-1 for MODY4 (9), and the HNF-1ß for MODY5 (10),
respectively. The most commonly identified cause of MODY in most racial
groups is a mutation in the HNF-1 gene (MODY3). Although the
mechanism of hyperglycemia in MODY3 is not fully understood, the
phenotypic characterization of MODY3 families has shown a deficient
insulin secretory response to glucose (11, 12). Because of the early
age of onset, the severe hyperglycemia, and the deficient insulin
secretion of patients with MODY3, we hypothesized that some patients
classified as having type 1B diabetes could have MODY3. In this study
we screened autoantibody-negative Japanese patients with type 1
diabetes at disease onset for mutations in the HNF-1 gene and
analyzed the functional properties of mutant genes.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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From a large population-based sample from patients with type 1 diabetes all of the antiislet autoantibody-negative, unrelated, new-onset patients (n = 28; 15 males and 13 females) were selected from 231 (92 males and 139 females; median age of onset, 17.0 yr) type 1 diabetic patients screened for antiislet autoantibodies. Their median age at onset and body mass index were 14.0 yr (range, 0.1–59.0 yr) and 18.8 kg/m2 (range, 13.1–22.8 kg/m2), respectively. They were all clinically diagnosed as having type 1 diabetes according to WHO criteria and required insulin therapy from the time of diagnosis (13). Two and 5 patients had at least 1 family member with type 1 and type 2 diabetes, respectively. Seventy-one percent (20 of 28) of these patients had DQA1*0301/DQB1*0303, DQA1*0301/DQB1*0401, or both, which are the high risk human leukocyte antigen (HLA)-DQ haplotypes for type 1 diabetes in Japanese populations (14). Sera were obtained from patients within 2 weeks after beginning insulin treatment.
An additional 54 patients with type 1A diabetes (23 males and 31 females) and 64 healthy control subjects (35 males and 29 females) were screened for those mutations that were found in autoantibody-negative patients with type 1 diabetes. The median age at onset in patients with type 1A diabetes was 19.5 yr (range, 3.0–59.0 yr), and 83% (45 of 54) were positive for GAD autoantibody, 52% (28 of 54) were positive for ICA512/IA-2 autoantibody, and 41% (22 of 54) were positive for phogrin (phosphatase homolog of granules of insulinoma)/IA-2ß autoantibody, respectively. All subjects gave informed consent, and the protocol was approved by the institutional review boards of the Nagasaki University School of Medicine. Sera were stored at -20 C until use.
Mutation analysis
The 10 exons, flanking introns, and the minimal promoter (the
354 bp upstream from the start codon) of the HNF-1 gene were
amplified by PCR using genomic DNA obtained from peripheral blood and
sequence-specific primers (15). PCR was performed in a 100-µL volume
containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1 mmol/L
MgCl2, 200 µmol/L deoxy-NTPs, 1 µmol/L of
each primer, 0.25 U AmpliTaq Taq polymerase
(Perkin-Elmer Corp., Foster City, CA), and 50 ng DNA. The
cycling conditions were 1 min at 94 C, followed by 35 cycles consisting
of 1 min at 94 C, 1 min at 60 C, and 1 min at 72 C. The PCR products
were purified using a Microcon-100 (Amicon, Inc., Beverly, MA) before
both strands were sequenced using a BigDye Terminator Cycle Sequencing
Ready Reaction Kit (Perkin-Elmer Corp. PE Applied Biosystems). The reactions were analyzed on an ABI Prism 377 DNA
Sequencer. The sequence of each mutation was confirmed by cloning the
PCR product into pGEM-T vector (Promega Corp., Madison,
WI) and sequencing clones representing both alleles. In the screening
for specific mutations in MODY family members, patients with
immune-mediated type 1 diabetes, and healthy control subjects, the
nucleotide substitution of GC to AA in the promoter region was
determined by PCR restriction fragment length polymorphism with the
restriction enzyme Fnu4HI. The
Pro379fsdelCT mutation was detected by a rapid
screening technique using fluorescently labeled forward primer
(5'-TCCCCTCGTAGGTCTCACGCAG-3') and modified reverse primer
(5'-GTTTCCAGGAAGTGAGGCCATCATG), generating a 137-bp fragment in
nonmutated samples and a 135-bp fragment from mutation alleles (16).
The PCR products were analyzed on a 4% polyacrylamide denaturing gel
on an ABI Prism 377 DNA Sequencer, and the difference in length between
normal and mutant alleles was detected by Genescan Analysis software
(ABI Perkin-Elmer Corp.).
Autoantibody assays
Autoantibodies to GAD were analyzed using RIA kits with 125I-labeled native GAD purified from pig brain (RIP anti-GAD Hoechst, Hoechst-Behring, Tokyo, Japan) as previously described (17). The insulin autoantibody assay was performed using a fluid phase radioassay with competition with cold insulin and precipitation with polyethylene glycol as previously described with some modifications (17). Autoantibodies to protein tyrosine phosphatase-like proteins, ICA512/IA-2 and phogrin/IA-2ß, were analyzed by radioassay using in vitro translated 35S-labeled ICA512bdc (amino acids 256–979 of IA-2) and the cytoplasmic domain of phogrin (amino acids 640-1015), respectively (18). The cut-off value was 5 U/mL for GAD autoantibodies, 80 µU/mL for insulin autoantibody assay, index 0.023 for ICA512/IA-2 autoantibodies, and index 0.031 for phogrin/IA-2ß autoantibodies, respectively. We participated in the international autoantibody proficiency programs for autoantibodies to GAD, insulin, and ICA512/IA-2 using these assays, and laboratory sensitivity and specificity for each assay were 100% and 100%, respectively.
Preparation of DNA constructs and functional study of the mutant
HNF-1 gene
A human HNF-1 complementary DNA (cDNA) clone including the
entire coding region in expression vector that has the cytomegalovirus
promoter was provided by Dr. Y. Yamada (Kyoto University School of
Medicine, Kyoto, Japan). The Pro379fsdelCT
mutation was introduced by the Sculptor in vitro mutagenesis
kit (Amersham International, Aylesbury, UK). Three
kilobases of human albumin (ALB) gene promoter containing the
HNF-1-binding site (-1773 to -1789, -358 to -342, and -65 to
-49 relative to the cap site) originally inserted in pBR-CAT vector
(19) were subcloned into the HnidIII site of the pGL3-Basic
vector (Promega Corp.). Ninety-two base pairs of the
HNF-1 gene promoter (+11 to +102 relative to the transcriptional
start site), which includes the wild-type (WT) and mutant activating
protein-1 (AP-1)-binding site, were amplified by PCR using genomic DNA
from the proband in family B and subcloned into a pGL3-Basic reporter
vector (Promega Corp.). The sequences of the constructs
were confirmed on an ABI Prism 377 DNA Sequencer.
HeLa cells were transfected with the indicated amounts of expression
and reporter vectors together with 50 ng pRL-simian virus 40 (SV40)
vector (Promega Corp.) as an internal control using
SuperFect transfection reagent (QIAGEN, Tokyo, Japan). The
trans-activation activities of WT-HNF-1 and
Pro379fsdelCT-HNF-1 were measured after 48 h using
the Dual Luciferase Reporter Assay System (Promega Corp.)
and TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). To analyze
the effect of the AP-1-binding site mutation +45/46 GC to AA, MIN6
cells and HuH7 cells were transfected with 2 µg WT or mutant HNF-1
promoter-pGL3, and the trans-activation activities were
measured after 48 h. Each study was repeated three or four
times.
Statistical analysis
Results are expressed as the mean ± SD unless otherwise indicated. Data in a luciferase reporter assay were analyzed by unpaired t test. P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene mutations in patients with type 1
idiopathic diabetes
Mutations were identified in 2 (7.1%) of 28 unrelated patients
with type 1 diabetes who lacked antiislet autoantibodies at disease
onset by sequencing of the 10 exons, flanking introns, and the minimal
promoter of the HNF-1 gene. The deletion of CT at codon 379 for Pro
(CCT) in exon 6 (Pro379fsdelCT) that would be
expected to generate a truncated protein of 416 amino acids was
identified in 1 family. A novel 2-bp substitution at nucleotide +45 (G
to A) and nucleotide +46 (C to A) from the transcriptional start site,
which is located in an AP-1-binding site of the promoter region and is
conserved in the sequences of human, rat, mouse, and chicken HNF-1
(15, 20), was identified in another family. Both mutations were
identified in heterozygous form and were not identified in 64 unrelated
healthy control subjects (128 chromosomes) and 54 unrelated patients
with type 1A diabetes (108 chromosomes).
Clinical profiles of subjects with mutations and their family members
The diabetic proband with frameshift mutation
Pro379fsdelCT was a 27-yr-old woman who had a
body mass index of 20.5 kg/m2. Her diabetes was
initially noticed by urine glucose screening at school at 12 yr of age
and was diagnosed by a subsequent oral glucose tolerance test (OGTT).
The low insulin secretory responses to glucose [fasting immunoreactive
insulin (IRI), 30.1 pmol/L; 30 min IRI, 74.2 pmol/L) and
arginine (fasting IRI, 22.8 pmol/L; 30 min IRI, 67.8 pmol/L) were
observed at the onset of the disease. Insulin treatment was started 5
months after diagnosis. Thus, she was initially diagnosed as a
slow-onset patient with type 1 diabetes. The amount of exogenous
insulin required was rapidly increased to 0.98–1.25 U/kg·day at 15
yr of age. She is a homozygote of HLA-DQA1*0301/DQB1*0401, which is one
of the highest risk haplotypes of type 1 diabetes in Japanese
population. She has severe diabetic retinopathy, with vitreous
hemorrhage and neovascular glaucoma and diabetic neuropathy. The
Pro379fsdelCT mutation was also identified in her
brother, father, and two paternal cousins from whom the genomic DNA was
able to be obtained (Fig. 1
, family A).
The proband, two aunts, and two cousins examined had overt diabetes,
and the father had impaired glucose tolerance as measured during an
OGTT. The brother showed normal glucose tolerance with impaired insulin
secretory response (fasting IRI, 13.4 pmol/L; 30 min IRI, 106.9 pmol/L)
at the time of examination. None of the examined subjects carrying the
mutation had autoantibodies to GAD, ICA512/IA-2, or
phogrin/IA-2ß.
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, family B).
DNA polymorphisms in the HNF-1 gene
In addition to the diabetes-associated mutations described above,
we found 11 nucleotide substitutions, of which 4 were located in exons
and 7 in introns (Table 1). All of these
have been described previously and were not associated with MODY (8, 21). Seven polymorphisms, including Ala98
Val,
Gly288, GGGGGC, Thr515,
ACGACA, and Gly574, GGCAGC; intron 1
nucleotide -91, AG, intron 5 nucleotide -47, CT, and intron 9
nucleotide +44, CT, which have been reported in whites and
African-Americans (8, 21, 22), were not found in our Japanese
subjects.
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gene
To analyze the functional properties of the
Pro379fsdelCT mutant HNF-1, a luciferase
reporter assay was performed using human cervical carcinoma HeLa cells,
which do not have endogenous HNF-1 (23). HeLa cells were transfected
with constructs encoding WT-HNF-1 and
Pro379fsdelCT-HNF-1 together with the human
ALB promoter-luciferase reporter gene. The mutant HNF-1 did not
stimulate transcription of the ALB-luciferase reporter, whereas
WT-HNF-1 stimulated transcription and generated a significant
increase in reporter gene activity (Fig. 2A). Increasing amounts of the
Pro379fsdelCT-HNF-1 inhibited luciferase
activity up to 53% of the control value in a dose-dependent manner,
suggesting that Pro379fsdelCT has a dominant
negative effect on HNF-1 activity (Fig. 2B).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene is characterized by rapid
progress to overt diabetes in childhood or adolescence and severe
insulin secretory defects in response to glucose (12, 26, 27).
We report here identification of the mutations in the HNF-1 gene in
2 of 28 antiislet autoantibody-negative patients with type 1 diabetes,
indicating that some of the MODY3 patients treated with insulin would
be misclassified as having type 1 diabetes. The results highlight the
difficulties in distinguishing between insulin-dependent diabetic
patients with MODY3 and patients with type 1B diabetes because of
the lack of antiislet autoantibodies in these patients (12, 28, 29).
The frameshift mutation Pro379fsdelCT identified
in exon 6 of the HNF-1 gene would generate a mutant truncated
protein of 416 amino acids, which affects the COOH-terminal
trans-activation domain of HNF-1 (30). The functional
studies indicate that this frameshift mutation has no
trans-activation activity and acts in a dominant negative
manner. Sourdive and co-workers reported that loss of residues 416–474
of HNF-1 not only impaired trans-activation but also
abolished nuclear transportation of the HNF-1 protein (31),
suggesting that this mutant is also lacking in proper nuclear targeting
signals. The mutation was present in five subjects; three were
diabetic, one had impaired glucose tolerance, and one had normal
glucose tolerance. The father in this family carried the mutation,
although he was nondiabetic at the age of 66 yr, indicating incomplete
penetrance.
The novel mutation in the promoter region (GCAA) at nucleotides +45
and +46 from the transcriptional start was found in the other nuclear
family. This mutation disrupts the binding site for the AP-1, which has
been shown to be important in activating the transcription factor
HNF-4 gene (32). The functional studies using MIN6 cells and HuH7
cells suggest that the +45/46 GC to AA mutation could lead to reduced
promoter activity of HNF-1 gene and thus might lead to lower than
normal levels of HNF-1 protein expression and consequently decreased
transcription of target genes encoding proteins that play a key role in
the insulin secretory response to glucose.
Moller and co-workers recently reported that in Danish patients with
type 1 diabetes without one of the high risk HLA haplotypes, DR3 or
DR4, about 10% of the patients carried a mutation in the HNF-1 gene
(29). However, both patients with type 1B diabetes carrying the
HNF-1 mutation reported here had HLA-DQA1*0301/DQB1*0401, a
haplotype that is a high risk HLA haplotype in Japanese subjects for
type 1 diabetes (14). Therefore, to avoid the misclassification of
MODY3 patients as type 1 diabetics, in whom an absolute deficiency of
insulin secretion is due to pancreatic ß-cell destruction, clinically
type 1 diabetic patients without antiislet autoantibodies should be
examined for mutations in the HNF-1 gene even if they have high risk
HLA haplotypes for type 1 diabetes.
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Acknowledgments |
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. Received July 14, 1999.
Revised September 15, 1999.
Accepted September 23, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene in maturity-onset diabetes
of the young. Nature. 384:458–460.[CrossRef][Medline]
gene in maturity-onset
diabetes of the young. Nature. 384:455–458.[CrossRef][Medline]
gene in MODY and
early-onset NIDDM: evidence for a mutational hotspot in exon 4. Diabetes. 46:528–535.[Abstract]
frameshift mutations; prevalence in maturity-onset diabetes
of the young and late-onset non-insulin dependent diabetes. Hum Genet.
351–354.
binding site in the promoter of the
hepatocyte nuclear factor-1 gene. Diabetes. 46:1648–1651.[Abstract]
gene in Danish
Caucasians with late-onset NIDDM. Diabetologia. 40:473–475.[CrossRef][Medline]
is a marker of atypical diabetes mellitus in African-American
children. Diabetologia. 42:380–381.[CrossRef][Medline]
gene in Japanese subjects with IDDM. Diabetes. 46:1643–1647.[Abstract]
gene in Caucasian families originally classified as having type I
diabetes. Diabetologia. 41:1528–1531.[CrossRef][Medline]
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, WT-HNF-1