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Steno Diabetes Center (L.H., S.U., J.N.J., T.H., O.P.) and Hagedorn Research Institute (H.V.P., P.S.), DK-2820 Gentofte, Denmark; Institute of Human Genetics, Panum Institute, University of Copenhagen (H.E.), DK-2200 N Copenhagen, Denmark; and Laboratory of Molecular Pathology of Diabetes, H. San Raffaele Scientific Institute (F.B.), I-20132 Milan, Italy
Address all correspondence and requests for reprints to: Lars Hansen, M.D., Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 2–6, DK-2820, Gentofte, Denmark.
Abstract
Increasing evidence suggests that defects in genes encoding
transcription factors that are expressed in the pancreatic ß-cells
may be important contributors to the genetic basis of type 2 diabetes
mellitus. Maturity-onset diabetes of the young (MODY) now exists in
five subtypes (MODY1–5), four of which are caused by mutations in
transcription factors hepatocyte nuclear factor-4
(HNF-4),
HNF-1, insulin promoter factor-1 (IPF-1), and HNF-1ß (MODY1, -3,
-4, and -5). Recent evidence from the British population even suggested
that IPF-1 may be a predisposing gene for type 2 diabetes. Thus,
highlighting the potential role of this transcription factor in the
genetic basis of Danish and Italian MODY as well as in Danish patients
with late-onset type 2 diabetes mellitus, we have examined the human
IPF-1 gene for mutations by single strand conformation polymorphism and
heteroduplex analysis in 200 Danish patients with late-onset type 2
diabetes and in 44 Danish and Italian MODY patients. In the patients
with late-onset type 2 diabetes we identified a noncoding G
insertion/deletion polymorphism at nucleotide -108, a silent G54G, and
a rare missense D76N variant. Moreover, a Danish MODY patient was
carrier of an A140T variant. Neither the D76N nor the A140T segregated
with diabetes, and their transcriptional activation of the human
insulin promoter expressed in vitro was
indistinguishable from that of the wild type (115 ± 21% and
84 ± 12% vs. 100%). We conclude that variants in
IPF-1 are not a common cause of MODY or late-onset type 2 diabetes in
the Caucasian population, and that in terms of insulin transcription
both the N76 and the T140 mutations are likely to represent
functionally normal IPF-1 variants with no direct role in the
pathogenesis of MODY or late-onset type 2 diabetes mellitus.
TYPE 2 DIABETES mellitus is genetically a
heterogeneous disease with an expanding number of genes being
identified as causing type 2 diabetes on a monogenic basis, such as the
maturity-onset diabetes of the young (MODY) (1, 2, 3, 4). The recently cloned
human insulin promoter factor-1 (IPF-1) gene (5) is essential for the
normal development of both the endocrine and the exocrine pancreas, as
evidenced by agenesis of the pancreas in knockout mice lacking both
alleles (6) and in humans homozygous for a frame-shift deletion that
introduces a premature stop codon and a truncated protein (7). In its
heterozygous form this deletion is linked to MODY4 in one pedigree (8),
but no variants were found in either French or Japanese MODY patients
(9, 10). Although MODY has the diagnostic criteria of an autosomal
dominantly inherited disease with onset before the age of 25 yr and
hence points toward the involvement of strong mutant alleles, weaker
alleles of the same genes with lower degree of penetrance and acting
either as codominant or recessive alleles are hypothesized to also be
involved in the more common late-onset form of type 2 diabetes
mellitus. Indeed, this hypothesis has already been tested for the
glucokinase gene (11, 12) and for the hepatocyte nuclear factor-1
(HNF-1) and -4 (HNF-4) genes, but no mutations were found to
be associated with type 2 diabetes (13, 14, 15).
Interestingly, a recent study of the British population has
reported functional mutations in the IPF-1 gene (C18R, D76N, and R197H)
that, analyzed together, are associated with type 2 diabetes (16).The
human IPF-1 gene, therefore, deserves the same attention as the
glucokinase and HNF-1/4 genes concerning its potential
involvement in late-onset type 2 diabetes mellitus.
Thus, to examine the potential pathogenic implications of genetic variability in IPF-1, we analyzed genomic DNA from 200 patients with late-onset type 2 diabetes of Danish ancestry and 44 patients of either Danish or Italian ancestry with MODY who had already been excluded as MODY2/3 carriers.
Materials and Methods
Subjects
Mutational analysis was performed in 200 type 2 diabetic patients consisting of 110 men and 90 women, aged 67 yr (range, 43–87 yr), with a mean body mass index of 30.6 kg/m2 (range, 22.3–48.8) and reported disease onset at 55.7 yr (range, 27–75); 11% were diet treated, 83% were treated with diet and/or oral hypoglycemic agents, and 6% were treated with insulin. The subjects were recruited either from the Danish family resource bank at the Department of Human Genetics, University of Copenhagen (Copenhagen, Denmark), or from the out-patient clinic at Steno Diabetes Center (13, 14, 15). A subgroup of the studied patients (n = 82) had been selected as 2-generation families for studies of quantitative trait loci, and of these, 80% had at least 1 first degree relative with type 2 diabetes or impaired glucose tolerance. All patients with late-onset type 2 diabetes were Danish Caucasians by self report. The study also included 44 MODY patients fulfilling the diagnostic criteria of an autosomal dominantly inherited type 2 diabetes with onset before the age of 25 yr (8 Danish and 36 Italian patients). Before participation, the purpose and risks of the study were carefully explained, both vocally and in writing, and informed consent was obtained. The protocol was approved by the committee of ethics in Copenhagen County and was in accordance with the Helsinki declaration.
Genetic analyses
The IPF-1 was analyzed by single strand conformational
polymorphism (SSCP) and heteroduplex analysis at two different
experimental settings (17) in a primary gene scanning on genomic DNA.
The IPF-1 gene comprising two exons of both coding and noncoding
regions was PCR amplified as follows: exon 1 in three overlapping
segments (Table 1
), and exon 2 in two
overlapping segments, as previously described (5). SSCP variants were
sequenced as described previously (17), and as the majority of reported
mutations in the human IPF-1 gene are located in exon 1 (7, 8, 16), the
coding region of this exon was also sequenced on one strand.
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T140 and N76 were introduced in the wild-type IPF-1 cloned into cytomegalovirus 5 plasmid using QuickChange (Stratagene, La Jolla, CA) as previously described (18). During control sequencing of the generated IPF-1 clones, two artificially introduced missense mutants were also discovered in one wild-type clone, E163, and in one N76 clone, Q54/N76. All of the sequenced clones were subsequently analyzed in the trans-activation assay.
Trans-activation of the human insulin gene promoter
Human IPF-1 (10 ng), either wild-type or mutant, was transfected into 30,000 NIH-3T3 mouse fibroblasts (24-well dishes) together with the following: coactivators NeuroD/BETA2 (10 ng) and E47 (5 ng), Renilla as internal standard (6 ng), human insulin promoter construct carrying luciferase reporter gene (200 ng), cytomegalovirus 4 plasmid as a negative control (25 ng), and pBluescript as a carrier (250 ng), using Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s recommendations. After transfection, cells were allowed to express for 16–24 h before lysis, and luciferase/Renilla activities were analyzed using a dual luciferase/Renilla reporter kit (Promega Corp., Madison, WI). Each series of transfections was performed in duplicate.
Results
Late-onset type 2 diabetes
Mutational analysis of 200 patients with late-onset type 2
diabetes showed the previously reported G insertion/deletion
polymorphism at nucleotide position -108 upstream of the translation
start site (19), and 2 subjects carried a silent G54G (ggc/gga). These
variants were not further investigated. In 1 patient we found a D76N
variant. The patient carrying the D76N variant is a 80-yr-old woman
treated with sulfonylurea urea with known diabetes since the age of 67
yr, fasting blood glucose of 9.1 mmol/L (6.7 mmol/L, 
normal), and
corresponding serum C peptide of 300 pmol/L. Because of the apparently
low prevalence of this D76N variant in the Danish Caucasian population,
we could not perform an association study, and no family members were
available for further genetic studies.
MODY patients
We found a A140T variant in exon 2 in one of the MODY patients. She had developed diabetes at the age of 13 yr and was treated with oral hypoglycemic agents until a pregnancy at age 25 yr made insulin treatment indispensable. Unfortunately, we did not have access to DNA of her diabetic parents and brother (who became diabetic at 25 yr of age); however, we could not detect the A140T variant in four uncles/aunts on her father’s side with late-onset diabetes. Thus, it is likely that the A140T variant did not segregate with diabetes in this pedigree from her pedigree, even though we cannot exclude that the affected branch of the family is the maternal one and that the paternal siblings are phenocopies.
Trans-activation assays
Because of the low prevalence of the D76N and A140T variants and
the lack of informative families, we investigated the biological
function of the D76N, A140T, and wild-type variants of human IPF-1 by
comparing the trans-activational efficiencies of the
variants on a luciferase construct harboring nucleotides -333 to +114
of the human insulin promoter. Each of the IPF-1 variants transfected
into NIH-3T3 mouse fibroblasts together with its coactivators
NeuroD/BETA2 and E47 resulted in comparable activation of the insulin
promoter, as estimated by luciferase activities (Table 2). Interestingly, when we investigated
the activity of the artificial E163 mutant and Q54/N76 double mutant
they both displayed impaired activity (E163, 44.6 ± 9.5%;
Q54/N76, 49.5 ± 9.1%) compared with wild-type IPF-1. The
observed differences were not the result of unequal IPF-1 expression,
as all of the IPF-1 clones (wild type, T140, N76, E163, and Q54/N76)
expressed the protein at equal levels (data not shown), as estimated by
Western analysis and immunoblotting with anti IPF-1 antibody 253
(18).
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The genetic basis for the common form of late-onset type 2 diabetes remains unknown, although several different chromosomal loci have been identified by genome-wide scannings (20, 21, 22, 23, 24, 25). It appears, however, that these loci potentially harboring diabetes genes are generally different and dependent on the examined diabetic population. In the present study we selected IPF-1 as a candidate gene for both the common late-onset type 2 diabetes and MODY in Caucasians (excluded as being MODY2/3). This gene has previously been investigated in diabetic patients fulfilling the criteria of maturity-onset diabetes of the young (9, 10), in Japanese late-onset type 2 diabetic patients (19), and in exon 2 in Americans (5). Sequencing of this gene in British patients with early-onset type 2 diabetes revealed three different mutations (C18R, D76N, and R197H) that, when expressed in Nes2y cells, displayed significant reductions in transcriptional activity (16). The C18R and R197H variants were only found in diabetic patients, whereas a more common D76N was found both in patients with type 2 diabetes as well as in glucose-tolerant control subjects (16). Another P33T variant has been reported in an Italian patient with early-onset type 2 diabetes (26), but the biological significance of this variant remains unknown.
In the present study of the human IPF-1 gene in Danish patients with MODY or late-onset type 2 diabetes or Italian patients with MODY, we found the D76N variant in 1 of 200 patients with late-onset type 2 diabetes and a novel A140T variant in 1 patient from a Danish MODY family. No potentially important mutations were found in patients from the Italian MODY families. The N76 and T140 variants, however, had normal transcriptional activity when tested on the human insulin promoter driving a luciferase reporter gene in NIH-3T3 mouse fibroblasts. The data from these in vitro studies are in contrast to the 30% reduction in activity reported for the N76 variant when expressed in Nes2y cells, but they corroborate the genetic data from the Danish population, where the prevalence of the D76N variant in Danish patients with type 2 diabetes is even lower (0.5% vs. 1%) than that in British control subjects (16). It is not known how the same variant of a gene can behave so differently; possible explanations are likely to include differences in cellular expression systems and population stratifications or perhaps founder effects, as has previously been reported for MODY2 among British diabetic families (27).
In summary, we found that Danish and Italian patients with MODY or late-onset type 2 diabetes harbor noncoding polymorphisms at nucleotides -108 and G54G as well as rare missense variants (D76N and A140T) in the coding region of the IPF-1 devoid of any major effect on transcription in our transfection system. We conclude that the D76N and A140T variants are not a common cause of MODY or late-onset type 2 diabetes in the examined Caucasian populations, and that in terms of insulin transcription both the N76 and T140 mutations are likely to represent functionally normal IPF-1 variants with no direct role in the pathogenesis of MODY or late-onset type 2 diabetes mellitus.
Acknowledgments
A special thanks to Violeta Stanojevic, Dorris Stoffers, and Joel Habener, Howard Hughes Medical Institute, Department of Molecular Endocrinology, Massachusetts General Hospital (Boston, MA), for their technical advice and generous supply of antibodies, IPF-1 clones, and human insulin promoter constructs. We also thank Helle Fjordvang, Annemette Forman, Lene Aabo, Ragna Jøgensen, and Bente Mottlau for dedicated and careful technical assistance, and Grete Lademann for secretarial support.
Footnotes
1 This work was supported in part by grants from the University of
Copenhagen, the Danish Medical Research Council, the Velux Foundation,
the Danish Diabetes Association, and the European Union
(BMH4-CT98-3084). 
Received September 10, 1999.
Revised November 9, 1999.
Accepted November 17, 1999.
References
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