A Novel eC192c/g Mutation in the Proximal P2 Promoter of the Hepatocyte Nuclear Factor-4 Gene (HNF4A) Associates With Late-Onset Diabetes
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糖尿病学杂志 2006年第6期
1 Steno Diabetes Center and Hagedorn Research Institute, Gentofte, Denmark
2 National Eye Clinic, Kennedy Institute, Glostrup, Denmark
3 Endocrinology Unit, Institut d’Investigacions Biomeediques August Pi i Sunyer, Hospital Clinic de Barcelona, Barcelona, Spain
4 Department of Pediatrics, Third Faculty of Medicine, Charles University, Prague, Czech Republic
5 Department of Medical Endocrinology M, Odense University Hospital, Odense, Denmark
6 Department of Obstetrics, Rigshospitalet, Copenhagen, Denmark
7 Research Centre for Prevention and Health, Glostrup University Hospital, Copenhagen, Denmark
8 Faculty of Health Science, University of Aarhus, Aarhus, Denmark
GDM, gestational diabetes mellitus; HNF, hepatocyte nuclear factor; IGT, impaired glucose tolerance; MODY, maturity-onset diabetes of the young; NGT, normal glucose tolerance; OGTT, oral glucose tolerance test
ABSTRACT
Recently, it has been shown that mutations in the P2 promoter of the hepatocyte nuclear factor (HNF)-4 gene (HNF4A) cause maturity-onset diabetes of the young (MODY), while single nucleotide polymorphisms in this locus are associated with type 2 diabetes. In this study, we examined 1,189 bp of the P2 promoter and the associated exon 1D of HNF4A for variations associated with diabetes in 114 patients with type 2 diabetes, 72 MODYX probands, and 85 women with previous gestational diabetes mellitus. A eC192c/g mutation was found in five patients. We screened 1,587 diabetic subjects and 4,812 glucose-tolerant subjects for the eC192c/g mutation and identified 5 diabetic and 1 glucose-tolerant mutation carriers (P = 0.004). Examination of the families showed that carriers of the eC192c/g mutation had a significantly impaired glucose-stimulated insulin release and lower levels of serum total cholesterol compared with matched control subjects. Furthermore, the mutation disrupted the binding of an unidentified sequence-specific DNA binding complex present in human islet extracts. Also, two novel linked polymorphisms in the P2 promoter at positions eC1107g/t and eC858c/t were identified. These variants were not significantly associated with type 2 diabetes or any pre-diabetic traits. In conclusion, a rare, novel mutation that disrupts a protein binding site in the pancreatic HNF4A promoter associates with late-onset diabetes.
Mutations in the coding region and the P2 promoter of the hepatocyte nuclear factor (HNF)-4 gene (HNF4A) cause maturity-onset diabetes of the young (MODY) (1eC3). Interestingly, studies in Caucasian populations have shown linkage between late-onset type 2 diabetes and the 20q region (4eC10), in which the HNF4A gene is located. Recently, a 90-kb genomic region covering the two HNF4A promoters, P1 and P2, which are located 46 kb apart, has been examined. In this region and close to the P2 promoter, several single nucleotide polymorphisms have been identified that associate with type 2 diabetes among both Ashkenazi Jewish subjects and Finns (11,12). These findings have been confirmed in the U.K. population, the Amish population, and the Danish population (13eC15). However, only a few studies have examined the HNF4A P2 promoter for variation associated with diabetes. We and others have previously identified mutations in the P2 promoter causing MODY or forms of early-onset diabetes (2,3,16). Furthermore, Mitchell et al. (17) have examined 25 MODYX probands and 48 early-onset type 2 diabetic patients for variants in 534 bp covering the minimal HNF4A P2 promoter and exon 1D. However, these authors failed to detect any diabetes-associated mutations.
In the present study, we have examined 1,189 bp of the P2 region and exon 1D of HNF4A for variation among different groups of noneCtype 1 diabetic patients. Furthermore, we tested for an association between identified gene variants and type 2 diabetes and/or intermediary pre-diabetic phenotypes.
RESEARCH DESIGN AND METHODS
The mutation screening was carried out on genomic DNA from 114 unrelated Danish type 2 diabetic patients of Caucasian origin (47 with diabetes diagnosed at 40 years of age), 85 women with previously diagnosed gestational diabetes mellitus (GDM) and with a family history of diabetes (at least one parent with diabetes), and 72 unrelated Caucasian MODYX patients from the Czech Republic and Denmark with clinical MODY (diabetes in at least two consecutive generations and at least one affected member of the family with onset before age 25 years) but without any known mutations in the genes encoding glucokinase (MODY2), HNF-1 (MODY3), and the P1 promoter and previous known exons of HNF4A (MODY1). All patients were recruited from the outpatient clinic at Steno Diabetes Center (Copenhagen), the Department of Obstetrics, National University Hospital (Copenhagen), the Department of Medical Endocrinology, Odense University Hospital (Odense), or the outpatient clinic at the Department of Pediatrics, Charles University (Prague). Diabetes was diagnosed in accordance with 1999 World Health Organization criteria. For the case-control study, 1,430 type 2 diabetic patients were recruited from the outpatient clinic at Steno Diabetes Center (n = 1,064 patients, including the 114 type 2 diabetic subjects included in the mutation screening) and the population-based Inter99 study, which was performed at the Research Centre for Prevention and Health (Glostrup, Denmark) (n = 366 patients). Control subjects comprised 4,812 individuals with normal glucose tolerance (NGT) and normoglycemia who were recruited from the population-based Inter99 study (n = 4,456) and randomly selected subjects from the Danish Central Population Register (n = 356) (18,19). The studies were approved by the local ethical committees of Copenhagen and Charles University (Prague) and were carried out in accordance with the principles of the Declaration of Helsinki II.
Mutation screening and genotyping.
The mutation screening was performed by either denaturing high-performance liquid chromatography (20) or single-stranded conformation polymorphismeCheteroduplex analysis (21). In our laboratory, these methodologies have an estimated sensitivity of >95% for detecting a variety of known mutations. The eC858 polymorphism was genotyped applying a mass spectrometryeCbased method as described (15), and the eC192 variant was genotyped by Taqman allelic discrimination (BioScience, Herts, U.K.).
Haplotypes.
The haplotypes were inferred with the expectation-maximization algorithm, using multiple starting points. Associations were tested using a general linear model implemented in the haplo.stats R-package (22).
Promoter functional analyses.
The eC192 variant was introduced in a luciferase reporter plasmid containing the P2 promoter sequence from positions eC371 to eC37 relative to the initiator codons (P2.371) and assessed by transient transfection analysis in MIN6 and CaCo cells as described (3).
Electrophoretic mobility shift assays.
Protein binding to the eC192 region of the P2 promoter was analyzed in nuclear extracts as previously described (23), except that the binding reaction contained 1 e蘥 poly(dI/dC), 10 mmol/l Tris HCl (pH 8.0), 0.5 mmol/l dithiothreitol, 1 e蘥/e蘬 BSA, 7% glycerol, and 2.5 mmol/l MgCl2.
Statistical analysis.
Fisher’s exact test was applied to test for significant differences in distribution of allelic frequencies or genotypes among type 2 diabetic patients and control subjects in the case-control study. All genotype distributions were tested for Hardy-Weinberg equilibrium using a likelihood ratio test. Differences in continuous variables between carriers of the polymorphisms were tested using a general linear model with age and BMI as covariates, sex and genotype as fixed factors, and familial structure as the random factor. SPSS for Windows (version 12.0) and SAS software were used for statistical analysis. A P value <0.05 was considered significant.
RESULTS
The mutation analysis revealed four nucleotide substitutions: a IVS1 +89g/a mutation, a eC192c/g mutation, and two polymorphisms (a eC1107g/t substitution and a eC858c/t substitution). The IVS1 +89g/a substitution was identified in one Czech MODYX patient. No cosegregation with diabetes was demonstrated (data not shown). The eC192c/g mutation was identified in a Danish MODYX proband, two type 2 diabetic patients, and two women with previously diagnosed GDM. Available family members (n = 19) were genotyped for the variant, and 8 additional mutation carriers were identified (Fig. 1). Furthermore, we screened for the variant among 4,812 glucose-tolerant Caucasian subjects and an additional 1,316 type 2 diabetic subjects and identified 1 additional mutation carrier, a 49-year-old glucose-tolerant woman. No family members of this mutation carrier were available for examination. Thus, we identified 1 glucose-tolerant mutation carrier among 4,812 control subjects and 5 diabetic mutation carriers among 1,587 unrelated diabetic subjects (1,430 type 2 diabetic subjects, 85 GDM women, and 72 MODYX subjects) (P = 0.004). Diabetic mutation carriers were on average diagnosed with diabetes at an age of 45.0 ± 15.5 years, slightly earlier than wild-type type 2 diabetic patients, who were diagnosed at an age of 52.2 ± 10.1 years.
The clinical and biochemical characteristics of the probands and their family members carrying the eC192c/g mutation, as compared with nonrelated subjects, are shown in Table 1. Subjects with NGT, impaired glucose tolerance (IGT), or type 2 diabetes were recruited from the population-based Inter99 study, which was performed at the Research Centre for Prevention and Health. For each eC192c/g mutation carrier, we selected 10 subjects matched on glucose tolerance status (NGT, IGT, or type 2 diabetes), sex, age, BMI, and fasting plasma glucose. The eC192c/g carriers had a low 30- and 120-min posteCoral glucose tolerance test (OGTT) serum insulin response (P = 0.005 and 0.02, respectively) and higher levels of plasma glucose at 120 min during an OGTT (P = 0.02) compared with matched subjects. Additionally, the eC192c/g carriers had significantly lower fasting serum total cholesterol (P = 0.01) (Table 1). When stratifying mutation carriers and matched control subjects into groups with NGT, IGT, and type 2 diabetes, we found that mutation carriers had a low 30- and 120-min post-OGTT serum insulin response in all subgroups (supplementary Tables 1eC3 [online appendix available at http://diabetes.diabetesjournals.org]).
Electrophoretic mobility shift assays showed that double-stranded oligonucleotides encompassing position eC192 of the P2 promoter exhibit sequence-specific, high-affinity binding to a single major nuclear complex that is not only enriched in human pancreatic islet nuclear extracts and INS1 -cells but also present in numerous other cell types (Fig. 2B and data not shown). Remarkably, the mutated sequence was unable to form a retardation complex and failed to compete with the complex formed with the wild-type sequence (Fig. 2B). However, transient transfection assays in cultured MIN6 and CaCo cells using a 334-bp P2 promoter luciferase reporter gene showed no significant impact of the mutation on basal or HNF-1eCdependent activity (data not shown).
The two upstream polymorphisms, at positions eC1107 and eC858, were in perfect linkage disequilibrium (R2 = 1); therefore we only made further analyses on the eC858 polymorphism. The allelic frequency of the eC858 polymorphism was 12.0% (95% CI 11.4eC12.7) among 4,812 control subjects and 11.8% (10.6eC13.0) among 1,430 type 2 diabetic patients (NS). Among 4,456 subjects with NGT and normoglycemia, there was a significant association between the polymorphism and alterations in fasting levels of serum total cholesterol and HDL cholesterol (P = 0.02 and 0.02, respectively) (Table 2). Association studies were also carried out using haplotypes based on single nucleotide polymorphisms identified and investigated in previous studies (15,18). However, there were no extended significant associations with diabetes or any relevant pre-diabetic trait for any combinations, including combinations of the eC858c/t polymorphism and the two previously reported diabetes-associated polymorphisms (rs1884614 and Thr130Ile) (data not shown) (15,18).
DISCUSSION
We report the identification of a eC192c/g mutation in the HNF4A P2 promoter that associates with diabetes. The mutation was identified among one MODYX proband, two type 2 diabetic patients, two women with previously diagnosed GDM, and one glucose-tolerant 49-year-old woman. The probands and the family members carrying the variant (n = 14) had a low serum insulin response during an OGTT compared with matched control subjects, indicating that they may have a pancreatic -cell defect. Their -cell function seems similar to what has been reported among two previously identified MODY mutations in the HNF4A P2 promoter (2,3) and is consistent with a decreased expression of HNF4A in pancreatic islets. Moreover, the fasting serum total cholesterol concentration of the eC192c/g mutation carriers was on average reduced by 18%. This finding may indicate that the expression of HNF4A in the liver is also decreased. Interestingly, a missense Thr130Ile HNF4A polymorphism has been described to be associated with decreased circulating total cholesterol levels (18). In contrast to what is typically seen with the majority of MODY1 mutations, the eC192c/g mutation does not seem to be highly penetrant. The ages for the diagnosis of diabetes varies between 25 and 75 years, and six mutation carriers have NGT or IGT at 35, 56, and 58 and 23, 33, and 49 years of age, respectively. Thus, our data indicate that the eC192c/g mutation is associated with late-onset familial diabetes. That the eC192c/g mutation can be associated with late-onset monogenic diabetes is further strengthened by a recent Norwegian study in which the eC192 mutation of HNF4A is shown to cosegregate with diabetes in two extended families (24).
The mutation did not affect basal and HNF-1eCinduced promoter activity in transient transfection assays in cell lines using a 334-bp reporter minigene. However, such an in vitro assay with a restricted DNA segment cannot by any means negate that this mutation may be functionally important in vivo in human pancreatic cells in the physiological chromatin and full genomic context. In favor of the idea that this mutation may indeed be functionally relevant for HNF4A gene activity is provided by the observation that it completely disrupts a high-affinity interaction site of the wild-type sequence with a human islet nuclear complex. This finding suggests that the eC192c/g mutation may disrupt a novel uncharacterized regulatory interaction in the HNF4A P2 promoter, which is analagous to two recently identified cis element mutations in this same segment of the P2 promoter causing MODY1 (Fig. 2A) (2,3). We therefore propose that the eC192c/g mutation may cause noneCtype 1 subtypes of diabetes through an abolished binding of a protein important in HNF4A gene regulation. However, in vivo functional assays will be required to conclusively test if this mutation can indeed alter the HNF4A gene transcription.
We identified two linked polymorphisms upstream of the transcription start site in the P2 promoter, eC1107g/t and eC858c/t, respectively. In a case-control study with a power of >80% to detect a relative risk of <1.2 with the observed allele frequency of 12%, we did not find an association with type 2 diabetes. Also, haplotype studies, including previously examined variants in the genomic region (15,18), did not reveal an association with type 2 diabetes. GenotypeeCquantitative trait studies in 4,456 subjects with NGT and normoglycemia suggested an association between the polymorphism and a reduced fasting level of serum total cholesterol and serum HDL cholesterol, indicating that these linked polymorphisms may affect the expression of HNF4A in the liver.
In conclusion, identified variations in the proximal P2 promoter of HNF4A do not contribute to the association to type 2 diabetes observed in this genomic region (11,12). However, a rare, novel eC192c/g mutation in the proximal HNF4A P2 promoter shown to affect binding of a human islet nuclear complex is associated with late-onset familial forms of diabetes.
ACKNOWLEDGMENTS
This study was supported by the Danish Diabetes Association; the Danish Medical Research Council; an EU grant, EUGENE2 (LSHM-CT-2004-512013); the Research Project of Czech MSM (0021620814); and the Juvenile Diabetes Research Foundation.
The authors thank Ds. H. Rder, L. Bjrkhaug, and P. Njlstad for sharing data and Drs. D. Bosco and T. Berney (University of Geneva) for human islets.
FOOTNOTES
J.E. and S.P.H. contributed equally to this study.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 National Eye Clinic, Kennedy Institute, Glostrup, Denmark
3 Endocrinology Unit, Institut d’Investigacions Biomeediques August Pi i Sunyer, Hospital Clinic de Barcelona, Barcelona, Spain
4 Department of Pediatrics, Third Faculty of Medicine, Charles University, Prague, Czech Republic
5 Department of Medical Endocrinology M, Odense University Hospital, Odense, Denmark
6 Department of Obstetrics, Rigshospitalet, Copenhagen, Denmark
7 Research Centre for Prevention and Health, Glostrup University Hospital, Copenhagen, Denmark
8 Faculty of Health Science, University of Aarhus, Aarhus, Denmark
GDM, gestational diabetes mellitus; HNF, hepatocyte nuclear factor; IGT, impaired glucose tolerance; MODY, maturity-onset diabetes of the young; NGT, normal glucose tolerance; OGTT, oral glucose tolerance test
ABSTRACT
Recently, it has been shown that mutations in the P2 promoter of the hepatocyte nuclear factor (HNF)-4 gene (HNF4A) cause maturity-onset diabetes of the young (MODY), while single nucleotide polymorphisms in this locus are associated with type 2 diabetes. In this study, we examined 1,189 bp of the P2 promoter and the associated exon 1D of HNF4A for variations associated with diabetes in 114 patients with type 2 diabetes, 72 MODYX probands, and 85 women with previous gestational diabetes mellitus. A eC192c/g mutation was found in five patients. We screened 1,587 diabetic subjects and 4,812 glucose-tolerant subjects for the eC192c/g mutation and identified 5 diabetic and 1 glucose-tolerant mutation carriers (P = 0.004). Examination of the families showed that carriers of the eC192c/g mutation had a significantly impaired glucose-stimulated insulin release and lower levels of serum total cholesterol compared with matched control subjects. Furthermore, the mutation disrupted the binding of an unidentified sequence-specific DNA binding complex present in human islet extracts. Also, two novel linked polymorphisms in the P2 promoter at positions eC1107g/t and eC858c/t were identified. These variants were not significantly associated with type 2 diabetes or any pre-diabetic traits. In conclusion, a rare, novel mutation that disrupts a protein binding site in the pancreatic HNF4A promoter associates with late-onset diabetes.
Mutations in the coding region and the P2 promoter of the hepatocyte nuclear factor (HNF)-4 gene (HNF4A) cause maturity-onset diabetes of the young (MODY) (1eC3). Interestingly, studies in Caucasian populations have shown linkage between late-onset type 2 diabetes and the 20q region (4eC10), in which the HNF4A gene is located. Recently, a 90-kb genomic region covering the two HNF4A promoters, P1 and P2, which are located 46 kb apart, has been examined. In this region and close to the P2 promoter, several single nucleotide polymorphisms have been identified that associate with type 2 diabetes among both Ashkenazi Jewish subjects and Finns (11,12). These findings have been confirmed in the U.K. population, the Amish population, and the Danish population (13eC15). However, only a few studies have examined the HNF4A P2 promoter for variation associated with diabetes. We and others have previously identified mutations in the P2 promoter causing MODY or forms of early-onset diabetes (2,3,16). Furthermore, Mitchell et al. (17) have examined 25 MODYX probands and 48 early-onset type 2 diabetic patients for variants in 534 bp covering the minimal HNF4A P2 promoter and exon 1D. However, these authors failed to detect any diabetes-associated mutations.
In the present study, we have examined 1,189 bp of the P2 region and exon 1D of HNF4A for variation among different groups of noneCtype 1 diabetic patients. Furthermore, we tested for an association between identified gene variants and type 2 diabetes and/or intermediary pre-diabetic phenotypes.
RESEARCH DESIGN AND METHODS
The mutation screening was carried out on genomic DNA from 114 unrelated Danish type 2 diabetic patients of Caucasian origin (47 with diabetes diagnosed at 40 years of age), 85 women with previously diagnosed gestational diabetes mellitus (GDM) and with a family history of diabetes (at least one parent with diabetes), and 72 unrelated Caucasian MODYX patients from the Czech Republic and Denmark with clinical MODY (diabetes in at least two consecutive generations and at least one affected member of the family with onset before age 25 years) but without any known mutations in the genes encoding glucokinase (MODY2), HNF-1 (MODY3), and the P1 promoter and previous known exons of HNF4A (MODY1). All patients were recruited from the outpatient clinic at Steno Diabetes Center (Copenhagen), the Department of Obstetrics, National University Hospital (Copenhagen), the Department of Medical Endocrinology, Odense University Hospital (Odense), or the outpatient clinic at the Department of Pediatrics, Charles University (Prague). Diabetes was diagnosed in accordance with 1999 World Health Organization criteria. For the case-control study, 1,430 type 2 diabetic patients were recruited from the outpatient clinic at Steno Diabetes Center (n = 1,064 patients, including the 114 type 2 diabetic subjects included in the mutation screening) and the population-based Inter99 study, which was performed at the Research Centre for Prevention and Health (Glostrup, Denmark) (n = 366 patients). Control subjects comprised 4,812 individuals with normal glucose tolerance (NGT) and normoglycemia who were recruited from the population-based Inter99 study (n = 4,456) and randomly selected subjects from the Danish Central Population Register (n = 356) (18,19). The studies were approved by the local ethical committees of Copenhagen and Charles University (Prague) and were carried out in accordance with the principles of the Declaration of Helsinki II.
Mutation screening and genotyping.
The mutation screening was performed by either denaturing high-performance liquid chromatography (20) or single-stranded conformation polymorphismeCheteroduplex analysis (21). In our laboratory, these methodologies have an estimated sensitivity of >95% for detecting a variety of known mutations. The eC858 polymorphism was genotyped applying a mass spectrometryeCbased method as described (15), and the eC192 variant was genotyped by Taqman allelic discrimination (BioScience, Herts, U.K.).
Haplotypes.
The haplotypes were inferred with the expectation-maximization algorithm, using multiple starting points. Associations were tested using a general linear model implemented in the haplo.stats R-package (22).
Promoter functional analyses.
The eC192 variant was introduced in a luciferase reporter plasmid containing the P2 promoter sequence from positions eC371 to eC37 relative to the initiator codons (P2.371) and assessed by transient transfection analysis in MIN6 and CaCo cells as described (3).
Electrophoretic mobility shift assays.
Protein binding to the eC192 region of the P2 promoter was analyzed in nuclear extracts as previously described (23), except that the binding reaction contained 1 e蘥 poly(dI/dC), 10 mmol/l Tris HCl (pH 8.0), 0.5 mmol/l dithiothreitol, 1 e蘥/e蘬 BSA, 7% glycerol, and 2.5 mmol/l MgCl2.
Statistical analysis.
Fisher’s exact test was applied to test for significant differences in distribution of allelic frequencies or genotypes among type 2 diabetic patients and control subjects in the case-control study. All genotype distributions were tested for Hardy-Weinberg equilibrium using a likelihood ratio test. Differences in continuous variables between carriers of the polymorphisms were tested using a general linear model with age and BMI as covariates, sex and genotype as fixed factors, and familial structure as the random factor. SPSS for Windows (version 12.0) and SAS software were used for statistical analysis. A P value <0.05 was considered significant.
RESULTS
The mutation analysis revealed four nucleotide substitutions: a IVS1 +89g/a mutation, a eC192c/g mutation, and two polymorphisms (a eC1107g/t substitution and a eC858c/t substitution). The IVS1 +89g/a substitution was identified in one Czech MODYX patient. No cosegregation with diabetes was demonstrated (data not shown). The eC192c/g mutation was identified in a Danish MODYX proband, two type 2 diabetic patients, and two women with previously diagnosed GDM. Available family members (n = 19) were genotyped for the variant, and 8 additional mutation carriers were identified (Fig. 1). Furthermore, we screened for the variant among 4,812 glucose-tolerant Caucasian subjects and an additional 1,316 type 2 diabetic subjects and identified 1 additional mutation carrier, a 49-year-old glucose-tolerant woman. No family members of this mutation carrier were available for examination. Thus, we identified 1 glucose-tolerant mutation carrier among 4,812 control subjects and 5 diabetic mutation carriers among 1,587 unrelated diabetic subjects (1,430 type 2 diabetic subjects, 85 GDM women, and 72 MODYX subjects) (P = 0.004). Diabetic mutation carriers were on average diagnosed with diabetes at an age of 45.0 ± 15.5 years, slightly earlier than wild-type type 2 diabetic patients, who were diagnosed at an age of 52.2 ± 10.1 years.
The clinical and biochemical characteristics of the probands and their family members carrying the eC192c/g mutation, as compared with nonrelated subjects, are shown in Table 1. Subjects with NGT, impaired glucose tolerance (IGT), or type 2 diabetes were recruited from the population-based Inter99 study, which was performed at the Research Centre for Prevention and Health. For each eC192c/g mutation carrier, we selected 10 subjects matched on glucose tolerance status (NGT, IGT, or type 2 diabetes), sex, age, BMI, and fasting plasma glucose. The eC192c/g carriers had a low 30- and 120-min posteCoral glucose tolerance test (OGTT) serum insulin response (P = 0.005 and 0.02, respectively) and higher levels of plasma glucose at 120 min during an OGTT (P = 0.02) compared with matched subjects. Additionally, the eC192c/g carriers had significantly lower fasting serum total cholesterol (P = 0.01) (Table 1). When stratifying mutation carriers and matched control subjects into groups with NGT, IGT, and type 2 diabetes, we found that mutation carriers had a low 30- and 120-min post-OGTT serum insulin response in all subgroups (supplementary Tables 1eC3 [online appendix available at http://diabetes.diabetesjournals.org]).
Electrophoretic mobility shift assays showed that double-stranded oligonucleotides encompassing position eC192 of the P2 promoter exhibit sequence-specific, high-affinity binding to a single major nuclear complex that is not only enriched in human pancreatic islet nuclear extracts and INS1 -cells but also present in numerous other cell types (Fig. 2B and data not shown). Remarkably, the mutated sequence was unable to form a retardation complex and failed to compete with the complex formed with the wild-type sequence (Fig. 2B). However, transient transfection assays in cultured MIN6 and CaCo cells using a 334-bp P2 promoter luciferase reporter gene showed no significant impact of the mutation on basal or HNF-1eCdependent activity (data not shown).
The two upstream polymorphisms, at positions eC1107 and eC858, were in perfect linkage disequilibrium (R2 = 1); therefore we only made further analyses on the eC858 polymorphism. The allelic frequency of the eC858 polymorphism was 12.0% (95% CI 11.4eC12.7) among 4,812 control subjects and 11.8% (10.6eC13.0) among 1,430 type 2 diabetic patients (NS). Among 4,456 subjects with NGT and normoglycemia, there was a significant association between the polymorphism and alterations in fasting levels of serum total cholesterol and HDL cholesterol (P = 0.02 and 0.02, respectively) (Table 2). Association studies were also carried out using haplotypes based on single nucleotide polymorphisms identified and investigated in previous studies (15,18). However, there were no extended significant associations with diabetes or any relevant pre-diabetic trait for any combinations, including combinations of the eC858c/t polymorphism and the two previously reported diabetes-associated polymorphisms (rs1884614 and Thr130Ile) (data not shown) (15,18).
DISCUSSION
We report the identification of a eC192c/g mutation in the HNF4A P2 promoter that associates with diabetes. The mutation was identified among one MODYX proband, two type 2 diabetic patients, two women with previously diagnosed GDM, and one glucose-tolerant 49-year-old woman. The probands and the family members carrying the variant (n = 14) had a low serum insulin response during an OGTT compared with matched control subjects, indicating that they may have a pancreatic -cell defect. Their -cell function seems similar to what has been reported among two previously identified MODY mutations in the HNF4A P2 promoter (2,3) and is consistent with a decreased expression of HNF4A in pancreatic islets. Moreover, the fasting serum total cholesterol concentration of the eC192c/g mutation carriers was on average reduced by 18%. This finding may indicate that the expression of HNF4A in the liver is also decreased. Interestingly, a missense Thr130Ile HNF4A polymorphism has been described to be associated with decreased circulating total cholesterol levels (18). In contrast to what is typically seen with the majority of MODY1 mutations, the eC192c/g mutation does not seem to be highly penetrant. The ages for the diagnosis of diabetes varies between 25 and 75 years, and six mutation carriers have NGT or IGT at 35, 56, and 58 and 23, 33, and 49 years of age, respectively. Thus, our data indicate that the eC192c/g mutation is associated with late-onset familial diabetes. That the eC192c/g mutation can be associated with late-onset monogenic diabetes is further strengthened by a recent Norwegian study in which the eC192 mutation of HNF4A is shown to cosegregate with diabetes in two extended families (24).
The mutation did not affect basal and HNF-1eCinduced promoter activity in transient transfection assays in cell lines using a 334-bp reporter minigene. However, such an in vitro assay with a restricted DNA segment cannot by any means negate that this mutation may be functionally important in vivo in human pancreatic cells in the physiological chromatin and full genomic context. In favor of the idea that this mutation may indeed be functionally relevant for HNF4A gene activity is provided by the observation that it completely disrupts a high-affinity interaction site of the wild-type sequence with a human islet nuclear complex. This finding suggests that the eC192c/g mutation may disrupt a novel uncharacterized regulatory interaction in the HNF4A P2 promoter, which is analagous to two recently identified cis element mutations in this same segment of the P2 promoter causing MODY1 (Fig. 2A) (2,3). We therefore propose that the eC192c/g mutation may cause noneCtype 1 subtypes of diabetes through an abolished binding of a protein important in HNF4A gene regulation. However, in vivo functional assays will be required to conclusively test if this mutation can indeed alter the HNF4A gene transcription.
We identified two linked polymorphisms upstream of the transcription start site in the P2 promoter, eC1107g/t and eC858c/t, respectively. In a case-control study with a power of >80% to detect a relative risk of <1.2 with the observed allele frequency of 12%, we did not find an association with type 2 diabetes. Also, haplotype studies, including previously examined variants in the genomic region (15,18), did not reveal an association with type 2 diabetes. GenotypeeCquantitative trait studies in 4,456 subjects with NGT and normoglycemia suggested an association between the polymorphism and a reduced fasting level of serum total cholesterol and serum HDL cholesterol, indicating that these linked polymorphisms may affect the expression of HNF4A in the liver.
In conclusion, identified variations in the proximal P2 promoter of HNF4A do not contribute to the association to type 2 diabetes observed in this genomic region (11,12). However, a rare, novel eC192c/g mutation in the proximal HNF4A P2 promoter shown to affect binding of a human islet nuclear complex is associated with late-onset familial forms of diabetes.
ACKNOWLEDGMENTS
This study was supported by the Danish Diabetes Association; the Danish Medical Research Council; an EU grant, EUGENE2 (LSHM-CT-2004-512013); the Research Project of Czech MSM (0021620814); and the Juvenile Diabetes Research Foundation.
The authors thank Ds. H. Rder, L. Bjrkhaug, and P. Njlstad for sharing data and Drs. D. Bosco and T. Berney (University of Geneva) for human islets.
FOOTNOTES
J.E. and S.P.H. contributed equally to this study.
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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