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Gene in a Danish Pedigree with Maturity-Onset Diabetes of the Young1
Steno Diabetes Center and Hagedorn Research Institute (A.M.M., L.T.D., L.A., L.H., T.H., O.P.), Gentofte, Copenhagen DK-2820, Denmark; and Department of Medicine M (Endocrinology and Diabetes) (O.S.), Kommunehospitalet, University Hospital of Aarhus, Aarhus D12-8000, Denmark
Address all correspondence and requests for reprints to: Ann Merete Møller, M.S., Steno Diabetes Center, Niels Steensens Vej 2, DK- 2820 Gentofte, Copenhagen, Denmark.
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Abstract |
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), glucokinase, HNF-1, insulin promoter factor-1,
and HNF-1ß genes] have been shown to cause maturity onset diabetes
of the young (MODY). About 50% of all known MODY in Danish Caucasian
MODY probands can be explained by mutations in the HNF-1 gene
(MODY3). To estimate the prevalence of MODY caused by mutations in the
HNF-4 gene (MODY1), we screened 10 non-MODY3 probands for mutations
in the minimal promoter and the 12 exons of the HNF-4 gene. One of
the probands had a novel frameshift mutation (Phe75fsdelT) in exon 2 of
the HNF-4 gene, resulting in a premature termination of translation
after 117 amino acids of the messenger RNA encoded by that allele. The
mutation cosegregated with diabetes in the pedigree and was not
detected in 84 unrelated Danish Caucasian healthy glucose-tolerant
control subjects or in 84 type 2 diabetic patients. At the time of
examination, 4 of 6 mutation carriers were treated with insulin and 2
with oral hypoglycemic medication. Two mutation carriers had
late-diabetic complications. Even though the HNF-4 protein is known
to be important in the regulation of genes involved in lipid
metabolism, carriers of the mutation did not differ from age and
sex-matched control subjects, in regard to levels of fasting serum
total cholesterol, serum high-density lipoprotein-cholesterol, and
serum triglyceride. In conclusion, by screening 10 non-MODY3 probands
for mutations in the HNF-4 gene, we identified 1 diabetes-associated
frameshift mutation (Phe75fsdelT), suggesting that defects in HNF-4
are a rare cause of MODY in Denmark. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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gene, MODY2 by mutations in the glucokinase
gene, MODY3 by mutations in the HNF-1 gene, MODY4 by mutations in
the insulin promoter factor-1 gene, and MODY5 is associated with
mutations in the HNF-1ß gene (1, 2, 3, 4, 5). About 50% of all known MODY
cases in Denmark can be explained by mutations in the HNF-1 gene
(6).
The HNF-4 protein is a highly conserved member of the nuclear
receptor superfamily of transcription factors and is expressed in the
liver, kidney, intestine, and pancreatic islets (7, 8). It is essential
in the regulation of the expression of several genes, including genes
involved in glucose, fatty acid and cholesterol metabolism, and in
liver differentiation (9, 10). Knockout of the HNF-4 gene in mice is
embryonic lethal, indicating that it is important for early development
(11). In humans, heterozygous carriers of a stop mutation, either at
codon 154 or at codon 268 of the HNF-4 gene, develop MODY (1, 12).
Functional experiments of the stop mutation at codon 268 (Q 268x)
have indicated that dimers of the encoded truncated proteins are unable
to bind DNA and that the mutated proteins may have a different
subcellular localization than the wild-type HNF-4 proteins (13). The
experiments also suggest that the pathogenic effect of the Q268X
mutation is caused by the reduced amount of functional HNF-4
proteins in carriers of the mutation, because the mutated proteins do
not inhibit the transactivating properties of the wild-type proteins
(13, 14). Furthermore, three missense mutations in the HNF-4 gene
have been shown to segregate with diabetes in MODY pedigrees (15, 16, 17). How the latter mutations cause diabetes is still unknown.
The present study was undertaken to examine the prevalence and the
nature of mutations in the HNF-4 gene in a subgroup of Danish MODY
probands without mutations in the HNF-1 gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Twenty unrelated Danish Caucasian MODY probands from families
where diabetes was diagnosed in at least one subject before the age of
25 yr, have previously been examined by direct sequencing for mutations
in the minimal promoter and the coding region of the HNF-1 gene (the
MODY3 gene). All the patients had a family history of diabetes that
occurred in at least three consecutive generations, consistent with an
autosomal dominant mode of inheritance. Ten non-MODY3 probands were
examined in the present study, for mutations in the HNF-4 gene.
Mutation analysis
PCR amplification of exon 1a, exon 1b, and exon 2–10 were performed using 100 ng of genomic DNA, as described previously (18). Exon 1c and the minimal promoter were amplified by specific primers: exon 1c: 5'-tgtaaaacgacggccagtttccag caaaagtcgatcc-3' and 5'-caggaaacagctatgaccctgaatcgctggagctggg-3'; segment 1 of the minimal promoter: 5'-tgtaaaacgacggccagtggggaactggaagggag-3' and 5'-caggaaacagctatgacccatgactcacgggcactc-3'; and segment 2 of the minimal promoter: 5'-tgtaaaacgacggccagtttggaggggtgggtgagtc-3' and 5'-caggaaacagctatgacccattctccctgcctccac-3'. The PCR amplifications of exon 1c and the minimal promoter were also performed using the PCR protocol described previously using 1.5 mmol/L (exon 1c) or 2.0 mmol/L (segment 1 and 2 of the promoter) MgCl2 in the PCR-buffer and an annealing temperature of 55°C in the PCR cycles (18). PCR products were purified using Microcon 100 microconcentrators (Amicon Inc., Beverly, MA) and sequenced using ABI PRISM DYE Primer (-21 M13 and M13 reverse) Cycle Sequencing Kit with Amplitaq DNA polymerase FS and an ABI 373 sequencer (PE Applied Biosystems, Foster City, CA).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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gene in one MODY proband. The deletion results in a
change of the reading frame downstream of codon 75 in the messenger RNA
transcribed from the mutated allele and a premature termination of
translation after 117 amino acids. The HNF-4 protein encoded by the
mutated allele will include the first activation domain and the DNA
binding domain of the HNF-4 protein but lack both the ligand binding
and dimerization domains, as well as the second activation domain (7).
The mutated protein could (in accordance with the functional studies of
the Q268X mutation) be presumed to lack transactivation activity (13, 14). The Phe75fsdelT mutation cosegregates with diabetes in the
examined family members (consistent with an autosomal dominant mode of
inheritance), and carriers of the mutation had diabetes diagnosed at
the age of 11–40 yr (Fig. 1
). The
diabetic patients had, at the time of examination, a body mass index
(BMI) between 21 and 32 kg/m2. Only one diabetic patient
was obese. He was diagnosed with diabetes at the age of 40 yr,
indicating that obesity had no major impact on the clinical onset of
diabetes. At the time of diagnosis, all patients were treated with
diet, oral hypoglycemic agents, or low doses of insulin (<10 U/day).
At the time of examination, four patients were treated with insulin and
two with oral hypoglycemic agents. Two of the patients have severe
diabetic complications. Subject II-2 has laser-treated proliferative
retinopathy and subject III-5 has end-stage renal failure and is blind,
because of proliferative retinopathy. Although the HNF-4 protein is
known to regulate genes involved in cholesterol and fatty acid
metabolism, carriers of the frameshift mutation did not differ from
age- and sex-matched control subjects [MODY patients (N = 4,
i.e. subjects II-2, III-3, IV-1, and IV-3) vs.
glucose tolerant control subjects (N = 242, 121 males, 121
females, aged 52 ± 14 yr)], regarding fasting serum total
cholesterol (5.3 ± 1.4 mmol/L vs. 5.5 ± 1.0 mmol/L),
serum high-density lipoprotein-cholesterol (1.4 ± 0.2 mmol/L
vs. 1.4 ± 0.4 mmol/L), and serum triglyceride levels
(1.0 ± 0.4 mmol/L vs. 1.2 ± 0.7
mmol/L)(mean ± SD) (9, 10).
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gene in the other 9 MODY probands did not reveal any genetic
variants, apart form previously identified common polymorphisms
(18).
Linkage studies have provided evidence of a major type 2 diabetic
susceptibility locus on chromosome 20q. However, one of the studies
suggests (together with a subsequent mutational scanning of the coding
region and minimal promoter of the HNF-4 gene) that linkage is not
caused by variants in the HNF-4 gene but by variants in another gene
in the same chromosomal region (19, 20, 21, 22, 23). Also, a previously reported
mutational screening of the HNF-4 gene in Danish Caucasian
late-onset type 2 diabetic patients excludes variants in the coding
region of the HNF-4 gene as a frequent cause of late-onset type 2
diabetes in the Danish Caucasian population (18). However, variants in
more distant regulatory domains than the minimal promoter or in introns
of the HNF-4 gene cannot be excluded as a cause of some cases of
type 2 diabetes and could possibly explain the positive linkage between
type 2 diabetes and a locus on chromosome 20q. Late-onset type 2
diabetes in one French pedigree has been shown to be caused by a
missense mutation (Val393Ile) in the HNF-4 gene. This mutation
cosegregates with late-onset type 2 diabetes and impaired insulin
secretion in the French pedigree and has, in functional experiments,
been shown to cause a reduced transactivation activity of the protein
in expression studies (24). Therefore, both early (MODY) and late-onset
forms of type 2 diabetes can be caused by mutations in the HNF-4
gene, though the prevalence of such forms of type 2 diabetes seems to
be low.
In summary, among 10 Danish Caucasian MODY probands without mutations
in the HNF-1 gene, a novel mutation, Phe75fsdelT, in the HNF-4
gene was identified. The mutation segregated with diabetes in the
pedigree and was not identified in either 84 Caucasian type 2 diabetic
patients with late onset or in 84 healthy Caucasian glucose-tolerant
control subjects. It seems that mutations in the HNF-4 gene are a
rare cause of MODY in Denmark.
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Acknowledgments |
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Footnotes |
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Received August 19, 1998.
Accepted October 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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gene in
maturity-onset diabetes of the young (MODY1). Nature. 384:458–460.[CrossRef][Medline]
gene in maturity-onset
diabetes of the young (MODY3). Nature. 384:455–458.[CrossRef][Medline]
gene. Diabetes. 46:726–730.[Abstract]
/MODY1 gene. J Clin Invest. 100:1400–1405.[Medline]
allows for
dimerization in solution but causes abnormal subcellular localization. Diabetes. 47:985–990.[Abstract]
regulates
expression of genes required for glucose transport and metabolism. Proc
Natl Acad Sci USA. 94:13209–13214./MODY1 gene and identification of a missense mutation,
R127W, in a Japanese family with MODY. Diabetes. 46:1652–1657.[Abstract]
in Caucasians with maturity onset NIDDM. Diabetologia. 40:980–983.[CrossRef][Medline]
as a candidate gene for
late-onset NIDDM linked with chromosome 20q. Diabetes. 47:970–972.[Medline]
, resulting in a reduced
transactivation activity, in human late-onset non-insulin-dependent
diabetes mellitus. J Clin Invest. 101:521–526.[Medline]
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