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Gene Mutations and Diabetes in Norway
Center for Medical Genetics and Molecular Medicine (L.B., A.M., P.R.N.), Haukeland University Hospital, and Department of Pediatrics (J.V.S., O.S., P.R.N.), University of Bergen, N-5021 Bergen; and Hormone Laboratory (P.T.), Aker University Hospital, N-0514 Oslo, Norway
Address all correspondence and requests for reprints to: Pål Rasmus Njølstad, M.D., Ph.D., Department of Pediatrics, University of Bergen, N-5021 Bergen, Norway. E-mail: pal.njolstad{at}uib.no.
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Abstract |
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gene cause maturity-onset diabetes of the young (MODY), type 3. To estimate the prevalence of MODY3 in Norwegian diabetic pedigrees, we screened a total of 130 families for HNF-1 mutations; 42 families with clinical MODY, 75 with suspected MODY, and 13 pedigrees with multiplex type 1 diabetes. Twenty-two families with clinical MODY, 15 families with suspected MODY, and one family with type 1 diabetes multiplex harbored HNF-1 mutations. Thus, in about half of Norwegian families with clinical MODY, mutations in the HNF-1 gene could be detected. Eight of the 18 different mutations identified were novel (G47E, T196fsdelCCAA, IVS3–1G>A, S256T, A276D, S445fsdelAG, M522V, and S531T). Haplotypes were determined for recurrent mutations, indicating a founder effect in Norway for the hot-spot mutation P291fsinsC and possibly also for P112L and R131W. To examine the molecular mechanisms underlying MODY3, we investigated the functional properties of 13 HNF-1 mutations. Two mutant HNF-1 proteins (R171X, R263C) were unable to bind DNA and at least five mutants (R131W, R171X, P379fsdelCT, S445fsdelAG, and Q466X) showed defective nuclear translocation. Transcriptional activation was reduced for most of the MODY3-associated mutants. Accordingly, the functional studies of HNF-1 mutants indicate that ß-cell dysfunction in MODY3 is caused by loss-of-function mechanisms like reduced DNA binding, impaired transcriptional activation, and defects in subcellular localization. |
Introduction |
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, HNF-1ß, HNF-4, insulin promoter factor-1, and neurogenic differentiation 1/ß-cell E-box transactivator 2 (1).
HNF-1 constitutes part of a network of transcription factors controlling organ-specific gene expression during embryonic development and in adult tissues. HNF-1 is expressed in the pancreatic ß-cell, and mutations in this gene lead to ß-cell dysfunction and diabetes mellitus (MODY3) in the heterozygous state. Diabetes mellitus in the homozygous state has been observed only for glucokinase/MODY2 (2) and insulin promoter factor-1/MODY4 (3). The presence of two defective HNF-1 alleles is assumed to be lethal in humans (4).
In some reports, MODY3 has been found to be the most frequent type of MODY, but others have observed that MODY2 is most common. This discrepancy may in part be explained by different ways of patient selection (5, 6). MODY3 is considered a serious type of diabetes because of a high frequency of late-diabetic complications (7, 8, 9, 10). Early diagnosis with appropriate treatment, however, seems to reduce the prevalence of chronic complications in patients with HNF-1 mutations (11). The clinical picture of MODY3 patients varies considerably, both within and between families, but the reasons for the differences are largely unknown. It has been proposed that the phenotypic expression of MODY3 results from either haploinsufficiency or a dominant negative effect of the mutant protein (1, 12, 13).
In a previous paper (14), we reported two novel loss-of-function HNF-1 mutations, P112L and Q466X, found in Norwegian MODY patients. In this study, we present a summary of all HNF-1 mutations and polymorphisms observed in subjects recruited from the Norwegian MODY Registry until May 2002. Because several, independently attained families harbored identical mutations, we investigated possible founder effects by determining haplotypes using markers in and near the HNF-1 locus. Finally, to obtain a better understanding of the molecular mechanisms underlying MODY3, we examined the functional properties of five novel and eight previously identified HNF-1 mutations by studying their effects on DNA binding, transcriptional activation, and subcellular localization.
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Subjects and Methods |
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A total of 117 families recruited from the Norwegian MODY Registry and 13 type 1 diabetes multiplex pedigrees were screened for mutations in HNF-1. The MODY Registry consists of patients for whom the referring physician suspected MODY, based on at least two of the following observations: first-degree relative with diabetes, early onset of diabetes (before 25 yr) inconsistent with type 1 diabetes, low insulin dosage requirement, or type 2 diabetes diagnosed before the age of 35 yr. Each family was then independently evaluated by P.R.N. and J.V.S., and 42 families were found to fulfill strict MODY criteria (autosomal dominant inheritance in two generations, at least two relatives of the proband having either diabetes or impaired glucose tolerance, onset of diabetes before 25 yr of age in at least one subject, and reduced insulin secretion). Seventy-five families had a suspected MODY diagnosis (limited clinical data available), including one family originating from Bangladesh and one female proband adopted from Colombia. The remaining families were of Caucasian ethnicity. In addition, 13 families, in which the proband and a first-degree relative had apparent type 1 diabetes (i.e. multiplex families), were included in the analysis. The reason for this inclusion was a previous study (15) that found about 10% type 1 diabetic families to be misclassified and harbor MODY3 mutations.
Patients were classified according to age of onset of diabetes, family history of the disease, treatment, and complications. The frequencies of -153InsTGGGGGT and M522V were assessed in a material of 100 healthy subjects (Norwegian blood donors). The studies were carried out in accordance with the Declaration of Helsinki and approved by the Regional Ethics Committee. Informed consent was obtained from all subjects.
Linkage analysis and screening for HNF-1 mutations
The families were typed with the microsatellite markers D12S366, D12S86, D12S321, and UC-39 using an ABI377 PRISM automated DNA sequencer and Gene Scan software (PE Applied Biosystems, Foster City, CA). Marker D12S76 and the intragenic marker HNF1A/M1 (forward primer: 5'-GTTCTGAGCTAGGACAGTTGG-3'; reverse primer: 5'-CTAGAACCTACTGAATCG-3') were added for the study of possible founder effects. For 13 families (all with clinical MODY), sufficient samples were available for linkage analysis. Of these, only families with an HNF-1 haplotype that cosegregated with the disease were scanned for mutations in this gene. In families with only one or two members available for analysis, the HNF-1 gene was sequenced directly. The exons, their flanking intronic regions, and the minimal promoter were PCR amplified and the products sequenced (16, 17). After finding an HNF-1 mutation in a proband, remaining family members were tested for the same mutation either by sequencing the exon in question or a specifically designed restriction enzyme-based test (18). Experimental conditions for the microsatellite analyses and the sequencing are available upon request.
Site-directed mutagenesis and construction of HNF-1 mutants for expression analysis
A construct encoding the human HNF-1 cDNA was prepared by cloning a cDNA fragment (nucleotides 1–2374; GenBank accession no. M57732) with an added polylinker in the 5'-end into the HindIII/EcoRI site of the expression vector pcDNA3.1+ (Invitrogen, Carlsbad, CA). The cDNA was also cloned in-frame into the BamHI/EcoRI site of vector pcDNA3.1/HisC (Invitrogen), which gives rise to proteins with an N terminal anti-Xpress antibody epitope. The mutant HNF-1 constructs were prepared by the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), using appropriate primers (primer sequences available upon request). A reporter gene construct, pGL3-RA, containing the promoter of the rat albumin gene (nucleotides -170 to +5) cloned into the SmaI site of the firefly luciferase reporter vector pGL3-Basic (Promega Corp., Madison, WI) was kindly provided by Professor Graeme Bell (Howard Hughes Medical Institute, University of Chicago, IL). The sequences of all constructs were confirmed by automated DNA sequencing.
DNA-binding studies
The DNA-binding assays were performed as described previously (14). In short, equivalent amounts of proteins, as determined by SDS-PAGE and immunoblotting (14), were incubated with 32P-labeled PE56 double-stranded DNA-fragment (5'-TGTGGTTAATGATCTACAGTTA-3') in a 25-µl reaction mixture containing 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% (vol/vol) glycerol, and 80 µg/µl sonicated salmon sperm DNA. DNA binding was quantified using the Luminescent image analyzer LAS-1000 Plus and program Image Gauge 3.12 (Fujifilm Medical Systems, Stanford, CA).
Cell culture and transfection studies
HeLa cells were cultured at 37 C in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml), and 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.). Transfection was performed using 3 µl FuGene6 (Roche Molecular Biochemicals, Indianapolis, IN) and a total of 2 µg DNA including 0.25 µg pcDNA3.1-HNF-1 or 0.25 µg pcDNA3.1-mutant HNF-1 (with or without epitope-tag), 0.50 µg of the reporter gene pGL3-RA, 1.225 µg empty vector (pcDNA3.1+ or pcDNA3.1/HisC), and 0.025 µg pRL-SV40 to control for efficiency of transfection. The transactivation activity was measured after 24 h using the dual-luciferase reporter assay system (Promega Corp.) and P values calculated by both the nonparametric Mann-Whitney test and unpaired t test using GraphPad Software Prism (GraphPad Software, Inc., San Diego, CA).
Subcellular localization of normal and mutant HNF-1
HeLa cells transfected with normal and mutant HNF-1 in pcDNA3.1/HisC were grown on poly-L-lysine-coated glass coverslips for 48 h. The immunofluorescence assay was performed as described previously (14) and 1:500 dilution of anti-X-press antibody in 5% donkey serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as primary antibody. The signal of the primary antibody was amplified by 1:500 diluted biotinylated antimouse IgG (Jackson ImmunoResearch Laboratories, Inc.) and streptavidin-conjugated Cy3 (Jackson ImmunoResearch Laboratories, Inc.). Nuclei were stained with Hoechst 33342 (Molecular Probes, Inc., Eugene, OR). Cy3 and Hoechst staining was visualized using an Eclipse E800M microscope (Nikon, Melville, NY) and appropriate filters.
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Results |
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for mutations and normal variants
Linkage analysis using microsatellite markers near the HNF-1 locus was performed in 13 families in which three or more subjects were available for investigation (Fig. 1
). These families had a strict clinical diagnosis of MODY. Four of these could be excluded because cosegregation of disease and an HNF-1 haplotype were not seen. In all nine families demonstrating linkage, HNF-1 mutations were identified by DNA sequencing. In families with only one or two subjects available, direct sequencing of HNF-1 was performed. Altogether, our screening revealed mutations in 38 of 130 families (Table 1), of which 37 pedigrees were clinical or suspected MODY families and one family had type 1 diabetes multiplex. The previously described frameshift mutation P291fsinsC in exon 4 (16, 19) was the most prevalent Norwegian MODY3 mutation and was identified in nine families (Table 1). Eight novel mutations were detected: G47E, T196fsdelCCAA, IVS3–1G>A, S256T, and A276D located in the DNA binding domain and S445fsdelAG, M522V, and S531T in the transactivation domain, respectively. Ten previously reported mutations, P112L, R131W, R171X, R229Q, R263C, R271W, P291fsinsC, P379fsdelCT, P447L, and Q466X (a proband from Colombia), were also observed (Table 1). The frequencies of all polymorphisms found during this study are given in Table 2.
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Among the 117 families in the MODY Registry, complete clinical data were available for most pedigrees. There were 37 different MODY3 families with altogether 79 mutation-carrying subjects. Four subjects were normoglycemic, six patients were diagnosed with impaired glucose tolerance (IGT), and 69 patients had diabetes. The age of diagnosis of diabetes was 20 ± 8 yr (range 6–54 yr, n = 60) (Table 3). Forty-seven subjects were diagnosed with diabetes before the age of 25 yr, and in 13 patients disease was recognized later. In nine diabetic MODY3 mutation carriers, the age of diagnosis was unknown. Body mass index was 22.4 ± 3.8 kg/m2 (n = 60). Fasting serum glucose in the diabetic MODY3 subjects was 8.2 ± 2.7 mmol/liter (n = 39). In the MODY3 individuals with IGT or normoglycemia, fasting serum glucose was 5.4 ± 0.7 mmol/liter and 4.5 ± 0.5 mmol/liter, respectively. HbA1c values in the three different groups were: 7.3 ± 1.4 (diabetic), 5.6 ± 0.4 (IGT), and 5.2 ± 0.3% (normoglycemic). Fasting serum insulin for diabetic MODY3 subjects was 103 ± 33 pmol/liter (n = 8) and 56 ± 35 pmol/liter (n = 15) (analyses performed at two different laboratories). Fasting serum C-peptide was 574 ± 249 pmol/liter (n = 19). Serum lipids were in the normal range; triglycerides, 1.25 ± 0.64 mmol/liter (n = 49); total cholesterol, 4.9 ± 1.2 mmol/liter (n = 50); high-density lipoprotein cholesterol, 1.4 ± 0.4 mmol/liter (n = 51); low-density lipoprotein cholesterol, 3.1 ± 1.1 mmol/liter (n = 35).
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Late-diabetic complications (retinopathy, nephropathy, and neuropathy) were seen in 13 MODY3 patients, but no information on complication status was available for eight subjects (Table 3
). Fifty-eight individuals did not have a history of any diabetes-associated complication. Nine patients were diagnosed with retinopathy, three patients were diagnosed with nephropathy, and a total of four subjects had neuropathy.
Inheritance pattern compatible with autosomal dominant inheritance was seen in 64 of 117 families from the MODY Registry. Of the 37 identified MODY3 families, autosomal dominant inheritance was reported in 27 pedigrees. Sufficient data to evaluate the insulin secretion (fasting serum insulin/C-peptide test alone or in addition to an oral glucose tolerance test) was available in subjects from 18 families only, of which reduced glucose stimulated insulin secretion was detected in individuals from 13 pedigrees. Probands from four families had normal fasting serum insulin, but one proband showed increased fasting serum insulin. Applying the strict criteria for MODY (see Subjects and Methods), including data on glucose-stimulated insulin secretion, 42 families had clinical MODY of which 22 families carried HNF-1 mutations (52%). Furthermore, 15 of the 75 families (20%) with suspected MODY (limited clinical data) had HNF-1 mutations.
Haplotype determination of families with HNF-1 mutations
Because several of the HNF-1 mutations were observed in independently collected families, it was of interest to examine whether the increased frequency could be attributed to a founder effect in the Norwegian population. To this end, microsatellite analysis using markers near and in the HNF-1 locus (see Subjects and Methods) was carried out for those families that shared a mutation and where haplotypes could be sorted out (Table 4). Evidence of identical or different haplotypes was confirmed by analyzing polymorphisms within the HNF-1 gene (data not shown).
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), more than one subject was available only in family N9. However, microsatellite determination performed on single subjects showed that allele sizes for the probands of N332 and N334 were consistent with the mutation-specific haplotype of N9. The single subjects from families N333 and N346 had allele sizes indicating that these two families could be related to each other but definitely not to N9. For the five families with the R131W mutation, haplotypes could be sorted out in three pedigrees (N7, N14, and N27). These haplotypes were, however, different from each other. The single subjects from the two remaining families (N13 and N94) had HNF-1 marker sizes consistent with the haplotype of family N14. Of particular interest was the haplotype pattern for the most frequent HNF-1 mutation in our material, namely P291fsinsC. In several Caucasian populations, this insertion has been observed in 20–30% of MODY3 families and is therefore denoted the MODY3 hot-spot mutation (13, 19, 20). Nine families, i.e. one fourth of the pedigrees with HNF1 mutation in the Norwegian MODY Registry, harbored the P291fsinsC mutation. Haplotypes could be unambiguously determined for families N1, N6, and N26. Except for some haplotype breaks occurring for the most distal markers, our data showed that these three pedigrees had mutant alleles segregating on a common chromosomal segment. Single subjects from families N29, N64, N69, and N75 exhibited genotypes consistent with the N1/N6/N26 haplotype, but N84 did not. The ninth P291fsinsC-positive family, N28, had immigrated to Norway from Bangladesh. Not surprisingly, the microsatellite analysis of the single available subject indicated an independent origin of the P291fsinsC mutation. Type 1 diabetes multiplex families
The novel mutation M522V was identified in 1 of 13 type 1 diabetes multiplex families. M522V was initially suspected to be a polymorphism but was not found in 100 healthy subjects. The proband, being a mutation carrier, had early-onset diabetes treated with insulin. Tests for antibodies against glutamic acid decarboxylase and protein tyrosine phosphatase-like molecule IA-2 were negative. Serum C-peptide concentrations were undetectable. A sister, also diabetic with detectable type 1 diabetes antibodies, did not have the mutation. The mother was healthy and not a mutation carrier. The father, however, harbored the mutation as well, but oral glucose tolerance test revealed a normal glucose response.
Subcellular localization of HNF-1 mutants
Subcellular localization of wild-type and mutant HNF-1 proteins in HeLa cells was investigated by immunofluorescence to analyze whether the mutations could affect nuclear localization. Localization of mutant proteins was studied in a minimum of 80 cells (Fig. 2 and Table 5). Wild-type HNF-1 was targeted to the nucleus in 95% of transfected cells. Cells transfected with mutants G47E, P112L, S256T, and M522V displayed an apparently normal pattern with protein staining confined to the nucleus in nearly all cells (>85%). Incorrect localization and accumulation of protein in both cytoplasm and nucleus (>85% of the cells) was observed for mutants R131W, R171X, P379fsdelCT, and Q466X. The immunofluorescent signals in cells transfected with R131W and R171X, though, were weaker and more difficult to evaluate. Mutants R229Q and R263C were targeted to the nucleus in the majority of the cells, but mutants R271W, A276D, and S445fsdelAG were targeted to both nucleus and cytoplasm in the majority of the cells. Cotransfecting the different mutants with wild-type HNF-1 increased the level of nuclear staining substantially for all mutants. However, cells cotransfected with Q466X and wild-type HNF-1 still demonstrated protein staining in the cytoplasm.
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proteins
DNA-binding ability of HNF-1 mutant proteins was examined by EMSA of in vitro translated proteins complexing with and binding the HNF1 site of a radioactively labeled double-stranded DNA-fragment (Fig. 3A). Binding was effectively competed by 100-fold excess of unlabeled double-stranded DNA-fragment (data not shown).
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protein. Addition of wild-type protein to the different mutants resulted in heterodimerization and complementation of poor DNA binding.
Transactivation potential of HNF-1 mutants
To assess the transactivation potential of wild-type and mutant constructs, we transfected HeLa cells, which lack endogenous HNF-1 expression. Stimulation of transcription was measured by luciferase reporter gene activity triggered by binding of HNF-1 to the rat albumin promoter (Fig. 3B).
Wild-type HNF-1 stimulated transcription by 9-fold. HNF-1 mutants P112L, R171X, R229Q, R263C, R271W, and Q466X exhibited significantly low levels of transcriptional activity (15–30% of wild type). The P379fsdelCT and R131W mutants displayed activities close to 50% of wild-type activity. No significant reduction in transcriptional activation could be demonstrated for mutants G47E, S256T, A276D, S445fsdelAG, and M522V. Cotransfecting cells with both wild-type and mutant proteins did not reduce wild-type activity significantly (Fig. 3B). The profiles shown represent nine experiments conducted over a period of 3 d.
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Discussion |
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To date, more than 120 different mutations in the HNF-1 gene have been identified in subjects of different ethnic backgrounds (21). Mutation analysis in 117 families recruited from the Norwegian MODY Registry revealed a MODY3 mutation in 37 pedigrees, and one mutation was identified among 13 type 1 diabetes multiplex families. The mutation P291fsinsC was the most prevalent, identified in nine MODY3 families. Six protein-truncating mutations were detected in our material, of which four were located in the transactivation domain. Two mutations were premature termination codon mutations (R171X, Q466X), and the four others were frameshift mutations (T196fsdelCCAA, P291- fsinsC, P379fsdelCT, S445fsdelAG). Among the 11 amino acid substitution mutations identified, eight were located in the DNA-binding domain. Nine of the 13 missense mutations were strictly conserved in HNF-1 sequences from man, mouse, chicken, frog, and salmon (R131W, R229Q, S256T, R263C, R271W, A276D, P447L, Q466X, and M522V). The nature and position of the HNF-1 mutations found in the Norwegian pedigrees are in accordance with the observation that mutations in the transactivation domain are likely to be protein truncating, whereas missense mutations predominate in the DNA-binding domain (22).
Clinical characteristics
Our study is in line with others showing that the majority of MODY3 subjects are diagnosed before the age of 25 yr. The diabetic MODY3 subjects had a relatively high fasting glucose. Several individuals demonstrated normal fasting serum insulin levels, but inappropriate insulin secretion, compared with the increased fasting serum glucose. This shows the importance of measuring both serum glucose and insulin/C-peptide in MODY3 patients when evaluating the insulin secretion status. The MODY3 subjects in our study had moderately elevated HbA1c-values (7.3% ± 1.4%).
In our study, we found that 22 families of the 42 pedigrees with a MODY phenotype had MODY3. Of 75 families, which were originally diagnosed with suspected MODY because of limited clinical data, 15 were found to have MODY3. This demonstrates the importance of the medical history of the proband and his family because a number of families could have been excluded for analysis if the clinical data had been more informative. The relatively lower number of MODY3 families in our study, compared with others (23), may be explained by increased number of other MODY subtypes in Norway, although this is not the case for MODY1, MODY2, and MODY6 (data not shown). Another reason could be differences in defining MODY based on clinical criteria. Moreover, the differential diagnosis between patients with MODY and young adult-onset diabetes can be difficult (23). Signs of the metabolic syndrome may be important markers (24) because body mass index and serum lipids in the Norwegian MODY3 subjects were in the normal range.
Founder effects in Norwegian families
Norway was severely hit by the Black Death about 25 generations ago. It has been estimated that two thirds of the Norwegian medieval population of about 500,000 was eradicated (25). After this genetic bottleneck, the number of inhabitants increased 30-fold to about 4.5 million over a time scale of less than seven centuries. During most of this period, population migration was hampered by geographical barriers and inefficient means of transportation. Until recently, the contribution from immigration to the population growth has been very limited.
One would therefore expect at least some of the recurrent Norwegian MODY3 mutations to be old and it should be possible to prove the founder effect by determining the haplotypes of the mutant alleles. This assumption was supported by our observation (based on information provided by the referring physicians) that families harboring identical HNF-1 mutations tended to live in the same regions of Norway. For the most common HNF-1 mutation, P291fsinsC, the microsatellite data suggested that in seven of nine families, the mutant allele had a common origin. Notably, one of the other P291fsinsC-positive families had emigrated from Bangladesh. This is the second time the HNF-1 hot-spot mutation has been reported in a MODY family of Asian ancestry (26), confirming the hypothesis that the P291 insertion has arisen independently several times in many different populations around the world (16, 19, 20, 27).
More enigmatic is the mutation P112L, which so far has been reported only from Norway and China (14, 28). The data from the microsatellite analysis (which were supported by studying internal HNF-1 polymorphisms found during the sequencing work; data not shown) are not straightforward to reconcile with a single origin of the mutation. Amino acid P112, encoded by CCG, might be more prone to change because CpG sites often are associated with mutational hot spots (29). Alternatively, the mutation could be very old, allowing enough recombination events to have occurred for P112L to be present on different genetic backgrounds today. Processes such as gene conversion may have contributed here.
The third, frequent MODY3 mutation in Norway is R131W, which also has been found in patients from North America and Great Britain (20, 28). The Norwegian R131W alleles seem to be present on three different chromosomal segments, again suggesting that the mutation is very old or it has arisen several times. In support of the latter possibility is the codon change (CGG to TGG), which affects a CpG site (see above). We further note that the mutation R131Q has been detected in at least five Caucasian and Japanese families (30), indicating that the R131 codon could be particularly susceptible to change. The observed genetic heterogeneity of R131W in Norway could also be due to contact with other populations because the country traditionally has had close connections to Great Britain and North America.
Subcellular localization of HNF-1 mutants
Import of nuclear proteins into the nucleus can be conferred by several distinct import signals, of which the classical nuclear localization signal (NLS) is best characterized. NLSs are generally short sequences that contain a high proportion of positively charged amino acids. To date, no single import signal in the HNF-1 protein has been identified, but there are three regions that fit the description of an NLS: region A (amino acids 158–171), region B (amino acids 197–205), and region C (amino acids 271–282). HNF-1 mutant R171X lacks region B and C because of a premature termination codon mutation. In accordance with earlier reports (31), R171X was localized both in the cytoplasm and nucleus. This suggests that loss of region B and C results in defective nuclear translocation. R271 is one of the positively charged amino acids forming the putative N-terminal cluster of the NLS-like region C of which A276 is also part. Disruption of this NLS-like region C by mutants R271W and A276D resulted in impaired nuclear targeting and accumulation of protein in the cytoplasm. The imperfect nuclear targeting observed for mutants P379fsdelCT (in accordance with earlier reports, Ref. 32) and Q466X (14) is in line with the fact that residues 441–474 have been implicated in normal transportation to the nucleus (33, 34) and loss of these amino acids may influence the process or rate of nuclear transport. Poor nuclear translocation was also observed for mutant R131W. Sequence alignments showed that R131 is a highly conserved residue in the HNF-1 and HNF-1ß genes of human, mouse, chicken, and frog. This amino acid could be important for the correct folding of the peptide chain, thereby indirectly affecting nuclear translocation.
DNA-binding ability of HNF-1 mutants
The DNA-binding domain of the HNF-1 gene consists of a dimerization domain (amino acids 1–32), a pseudo-POU-like domain (amino acids 100–172), and a homeodomain (amino acids 199–278). The pseudo-POU-like domain confers orientational specificity on homeodomain DNA binding (35). The R171X mutant, which lacks the essential homeodomain and transactivation domain because of a premature termination codon mutation, failed to bind DNA. These findings are in accordance with earlier reports (31). The poor DNA binding properties of P112L (14) and R131W could be a result of failure of mutant protein to properly orient itself on DNA binding because both mutations are positioned in the pseudo-POU-like domain. The complete absence of DNA binding for R263C (32) and the very weak binding for R229Q suggest both arginines to be essential to the homeodomain and the DNA-binding event because they are both highly conserved in HNF-1 and HNF-1ß genes of other species.
Transcriptional activation by HNF-1 mutants
The transactivation domain (amino acids 281–631) consists of two smaller domains: ADI (amino acids 546–628/Ser-rich) and ADII (amino acids 281–318/Pro-rich). ADI and ADII are required for efficient induction of liver-specific promoter activity (33). Mutant R171X lacks both ADI and ADII and demonstrated poor levels of transcriptional activity, probably because of complete lack of the transactivation domain. P379fsdelCT, S445fsdelAG, and Q466X, which lack ADI only, demonstrated somewhat higher levels of transcriptional activity than R171X. This observation is in agreement with previous studies (31, 32, 33) in which deletion of ADI reduced transcriptional activity to about 50% of wild-type activity, but deletion of ADII caused a more severe drop in activity, down to about 10% of the wild type.
The WFXNXR motif (amino acids 267–272) located in helix 3 of HNF-1 is strictly conserved among all known homeodomain sequences. It seems reasonable to believe that disruption of helix 3 by substituting amino acids could cause a conformational change in the protein, which again would affect protein function. R271, which is located in helix 3 and is part of the WFXNXR motif, is also strictly conserved in HNF-1 genes from fish to man. Substituting R271 with W271 resulted in a protein with low transactivation abilities. The reduction in transactivation for mutants P112L, R131W, R229Q, and R263C was expected, given their weak DNA-binding properties. Cotransfecting the different mutants with wild-type HNF-1 did not reduce wild-type activity significantly, which suggests that none of the 13 mutants function as dominant negative regulators.
MODY3-associated mutations or polymorphisms?
The missense mutations G47E and S256T were identified in families in which only one member was available for analysis. A determination of whether the mutations cosegregated with disease was therefore not possible. For the mutation M522V, samples of other family members were available. This mutation, which was found in the nondiabetic father of the proband was, however, not observed in his diabetic sister or detected in 100 healthy subjects studied. Although amino acids G47, S256, and M522 are strictly conserved in HNF-1 genes from man to fish, functional studies of the mutant proteins showed close to normal levels of activity. It is therefore conceivable that G47E, S256T, and M522V are normal but rare variants, not fully penetrant mutations, or risk factors for developing diabetes. This hypothesis should be investigated in complete family materials and by case/control studies.
Another interesting DNA variant is -153InsTGGGGGT, which was found in a proband with suspected MODY. This insertion has not previously been linked with the diabetic disease, but other mutations in the HNF-1 promoter have been associated with MODY3 (36). Because of the absence of other MODY mutations in the patient and low incidence of -153InsTGGGGGT in normal Norwegian controls (1/200 chromosomes) as well as in other Scandinavians (37), there is a possibility for the variant to predispose for the diabetic phenotype.
In conclusion, 52% of Norwegian families with clinical MODY, but only 20% of the patients with suspected MODY (as diagnosed by the referring physicians) had a mutation in the HNF-1 gene. Applying the classical MODY criteria is important in a genetic screening setting. A founder effect was likely for the three recurrent HNF-1 mutations P291fsinsC, R131W, and P112L. Functional analysis suggests that P112L, R131W, R171X, R229Q, R263C, R271W, A276D, P379fsdelCT, S445fsdelAG, and Q466X are loss-of-function mutations, but G47E, S256T, and M522V are either normal variants or mild mutations that might confer a risk for developing diabetes.
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Acknowledgments |
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Footnotes |
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Abbreviations: HNF, Hepatocyte nuclear factor; IGT, impaired glucose tolerance; MODY, maturity-onset diabetes of the young; NLS, nuclear localization signal.
Received June 24, 2002.
Accepted November 12, 2002.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-deficient mice. J Clin Invest 101:2215–2222[Medline]
/MODY3 gene in Japanese subjects with early- and late-onset NIDDM. Diabetes 46:1504–1508[Abstract]
diabetes (MODY3). Diabetologia 43:816–818[CrossRef][Medline]
is dominant negative. Diabetes 47:1231–1235[Abstract]
loss-of-function mutations (P112L and Q466X). Biochem Biophys Res Commun 279: 792–798
gene in Caucasian families originally classified as having type I diabetes. Diabetologia 41:1528–1531[CrossRef][Medline]
gene in maturity-onset diabetes of the young (MODY3). Nature 384:455–458[CrossRef][Medline]
gene hinders the identification of mutations in three families with maturity-onset diabetes of the young. Diabetes 48:921–923[Medline]
/MODY3 gene. Diabetologia 43:818–819[CrossRef][Medline]
gene in MODY and early-onset NIDDM: evidence for a mutational hotspot in exon 4. Diabetes 46:528–535[Abstract]
gene are a common cause of maturity-onset diabetes of the young in the U.K. Diabetes 46:720–725[Abstract]
gene in Japanese early-onset type 2 diabetes. Horm Metab Res 32:373–377[Medline]
mutations in MODY. Diabetologia 45:744–746[CrossRef][Medline]
(HNF-1 ) mutations in maturity-onset diabetes of the young. Hum Mutat 16:377–385[CrossRef][Medline]
, a diabetes-associated transcription factor. Biochem Biophys Res Commun 266:196–202[CrossRef][Medline]
(TCF1) promoter region in MODY patients. Hum Mutat 15:173–180[CrossRef][Medline]
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