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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2159
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 5 3054-3059
Copyright © 2005 by The Endocrine Society

The Functional Thr130Ile and Val255Met Polymorphisms of the Hepatocyte Nuclear Factor-4{alpha} (HNF4A): Gene Associations with Type 2 Diabetes or Altered ß-Cell Function among Danes

Jakob Ek, Christian Schack Rose, Dorit Packert Jensen, Charlotte Glümer, Knut Borch-Johnsen, Torben Jørgensen, Oluf Pedersen and Torben Hansen

Steno Diabetes Center and Hagedorn Research Institute (J.E., C.S.R., D.P.J., C.G., K.B.-J., O.P., T.H.), DK-2820 Gentofte, Denmark; Research Centre for Prevention and Health, Glostrup University Hospital (C.G., K.B.-J., T.J.), DK-2600 Glostrup, Denmark; and Faculty of Health Science, University of Aarhus (K.B.-J., O.P.), DK-8000 Aarhus, Denmark

Address all correspondence and requests for reprints to: Jakob Ek, M.Sc., Ph.D., Steno Diabetes Center and Hagedorn Research Institute, Niels Steensens Vej 6, NSK1.14, DK-2820 Gentofte, Denmark. E-mail: jaek{at}steno.dk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
HNF4A encodes an orphan nuclear receptor that plays crucial roles in regulating hepatic gluconeogenesis and insulin secretion. The aim of the present study was to examine two rare missense polymorphisms of HNF4A, Thr130Ile and Val255Met, for altered function and for association with type 2 diabetes (T2D). We have examined these polymorphisms 1) by in vitro transactivation studies and 2) by genotyping the variants in 1409 T2D patients and in 4726 glucose-tolerant Danish white subjects. When tested in COS7 cells, both the Thr130Ile and the Val255Met variants showed a significant decrease in transactivation activity compared with wild-type (73% of wild-type, P = 0.02, and 76%, P = 0.04, respectively). The Thr130Ile variant had a significantly increased carrier frequency among T2D patients compared with glucose-tolerant subjects [odds ratio, 1.26 (1.01–1.57); P = 0.04]. The rare Val255Met polymorphism had a similar frequency among T2D patients and glucose-tolerant subjects. Heterozygous glucose-tolerant carriers of the variant showed, however, decreased levels of fasting serum C-peptide (76%; P = 0.03) and decreased fasting serum triglyceride (58%; P = 0.02). In conclusion, The Thr130Ile and the Val255Met polymorphisms decrease the transcriptional activity of HNF4A, and the Thr130Ile polymorphism associates with T2D, whereas the Val255Met variant associates with a decrease in fasting serum C-peptide.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
STUDIES OF GLUCOSE-TOLERANT first-degree relatives of patients with type 2 diabetes (T2D) have shown impaired first- and second-phase insulin secretion, suggesting that defects in the pancreatic ß-cells are likely to be an important genetic predisposing factor in the pathogenesis of T2D (1). Also, prospective studies indicate that impaired insulin secretion is a predictor of T2D (2, 3). In line with these findings, several studies in humans and mice have indicated that defects in proteins known to regulate insulin secretion are the cause of monogenic subtypes of diabetes. The hepatocyte nuclear factor-4{alpha} gene (HNF4A) is expressed in liver, kidney, intestine, and pancreatic ß-cells. It is alternatively spliced creating several splice forms (4, 5, 6). The transcription of the gene is initiated by two different promoters, P1 and P2, located 46 kb apart, that drive the expression of HNF4A primarily in liver and ß-cells, respectively (7, 8, 9). The HNF-4{alpha} is a member of the nuclear hormone receptor family (10), and it functions as a homodimer interacting with promoter sequences of genes, which are regulated by HNF-4{alpha}. In the liver, HNF-4{alpha} is required for obtaining normal hepatic gluconeogenesis (11), whereas in the pancreatic ß-cells it is involved in regulating glucose metabolism as well as insulin expression and secretion (12, 13, 14). Several different mutations identified in the coding regions of HNF4A have been shown to be the cause of a specific form of maturity-onset diabetes of the young (MODY1) (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Functional studies of these mutations imply that they are loss-of-function variants, suggesting that MODY1 may emerge as a result of haploinsufficiency (16). Furthermore, two MODY-causing mutations have recently been identified in the P2 promoter of the HNF4A gene (8, 9).

Markers in the region flanking the HNF4A gene on chromosome 20q13 (22) have in family studies been linked to late-onset T2D among Caucasians (25, 26, 27, 28, 29). Recently, two independent studies have examined a 90-kb region covering both the P1 and P2 promoters of HNF4A and identified single-nucleotide polymorphisms (SNPs) that were associated with an increased risk of T2D (30, 31). This association could explain most of the identified linkage to T2D in the examined families (30, 31).

In a Danish study, T2D patients were examined for variation in the coding region of HNF4A in relation to common T2D (32), and two variants were identified (Thr130Ile and Val255Met). The minor allele frequency of the Thr130Ile among 509 T2D patients was 4.7% compared with 1.9% among 239 control subjects. However, this significant difference in minor allele frequency could not be reproduced in a Swedish case-control study (32). The Val255Met variant was exclusively identified in four of 477 T2D patients and in none of 217 matched control subjects (32). Thus, the aims of this study were 1) to examine in a larger study whether the two known coding polymorphisms of the HNF4A gene are associated with T2D or prediabetic traits in glucose-tolerant subjects and 2) to elucidate the potential functional impact of the two HNF4A polymorphisms by in vitro transactivation studies in COS7 cells.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

For the case-control study, 1466 T2D patients were recruited from the outpatient clinic at Steno Diabetes Center (SDC) between 1992 and 2001 (1094 patients) and from the population-based Inter99 study performed at the Research Centre for Prevention and Health between 1999 and 2001 (372 T2D patients) (33). Control subjects comprised 4520 glucose-tolerant individuals recruited from the population-based Inter99 study and 345 middle-aged subjects recruited at random through the Danish Central Population Register (Table 1Go). Only subjects with known normal glucose tolerance according to World Health Organization criteria were included as control subjects. Subjects with a known family history of diabetes were not excluded from the two control populations.


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  		TABLE 1. Characteristics of the examined Danish white individuals

 
All subjects who participated in the present protocol were living in the same area of Copenhagen. The Inter99 cohort is a population-based randomized nonpharmacological intervention study for prevention of cardiovascular disease undertaken at the Research Centre for Prevention and Health in Glostrup, Copenhagen County. Details of this cohort have been reported (33). Diabetes was diagnosed in accordance with the World Health Organization 1999 criteria. Clinical characteristics of the study groups included in the present investigation are given in Table 1Go.

There are a variable number of diabetic patients and glucose-tolerant control subjects in each study because of varying genotyping success rates of the two polymorphisms. Genotype-quantitative trait interaction studies were performed in 4391 (Thr130Ile) or in 4458 (Val255Met) population-based sampled glucose-tolerant subjects from the Inter99 study, who all had been examined by a standard 75-g oral glucose tolerance test (OGTT) with measurements of circulating levels of plasma glucose, serum insulin, and C-peptide at 0, 30, and 120 min.

All participants were Danish whites by self-report. Informed consent was obtained from all study participants before participation. The studies were approved by the ethical committee of Copenhagen and were in accordance with the principles of the Declaration of Helsinki II.

Genotyping

The samples were genotyped for the two SNPs (Thr130Ile and Val255Met) in HNF4A applying a mass-spectrometry-based method as described (34). The genotyping success rates were 97% for the Thr130Ile and 99% for the Val255Met variants, respectively. To elucidate the genotyping error rate, we examined a minimum of 88 replicate samples for each SNP, respectively, and among these there were no mismatches. The genotype distributions of both polymorphisms were in Hardy-Weinberg equilibrium.

Biochemical assays

Hemoglobin (Hb)A1C was analyzed by principles of ion-exchange HPLC using Bio-Rad VARIANT HbA1C (Bio-Rad, Hercules, CA) (normal range, 4.1–6.4%). Serum triglycerides, serum total cholesterol, and serum high-density lipoprotein (HDL) cholesterol were analyzed using enzymatic colorimetric methods (GPO-PAP and CHOD-PAP, Roche Molecular Biochemicals, Mannheim, Germany; and NEFA C, Wako, Neuss, Germany). The plasma glucose concentration was analyzed by a glucose oxidase method (Granutest; Merck, Darmstadt, Germany), and serum specific insulin (excluding des(31, 32)- and intact proinsulin) and serum C-peptide were measured by time-resolved fluoroimmunoassay (AutoDELFIA; Perkin-Elmer-Wallac, Turku, Finland).

Constructs

The transactivation studies were carried out applying a firefly renilla luciferase assay. We created a construct with full-length HNF4A cDNA cloned into the linker region of a pcDNA3 vector (Invitrogen AS, Taastrup, Denmark) (HNF4A-pcDNA3). Next, we generated two separate constructs with the HNF4A Thr130Ile and Val255Met mutations, respectively, in the HNF4A-pcDNA3 construct applying site-directed mutagenesis (QuikChange Site Directed Mutagenesis Kit; Stratagene, AH Diagnostic, Aarhus, Denmark) by use of the following specific primers: HNF4a130for, 5'-GGACCGGATCAGCATTCGAAGGTCAAGCT-3'; HNF4a130rev, 5'-AGCTTGACCTTCGAATGCTGATCCGGTCC-3'; HNF4a255for, 5'-CGGAGATGAGCCGGATGTCCATACGCATCC-3'; and HNF4a255rev, 5'-GGATGCGTATGGACATCCGGCTCATCTCCG-3' (substituted nucleotides compared with the wild-type sequence are shown in bold). The empty construct (pcDNA3) functioning as a control plasmid and pcDNA3 harboring wild-type HNF4A insert (HNF4A-pcDNA3) or inserts carrying the Thr130Ile or Val255Met variants were transformed into Epicurian Coli XL-2 Blue ultracompetent cells (Stratagene). The same procedure was followed for a reporter plasmid consisting of a pGL3 (Promega, Ramcon, Birkerød, Denmark) vector harboring the minimal promoter of HNF1A (nucleotides –361 to +48, relative to the transcription start site) upstream of the firefly luciferase gene. pcDNA3 and pGL3 plasmids were purified using the Maxi-prep (QIAfilter Plasmid Maxi Kit; QIAGEN, VWR International AS, Albertslund, Denmark) method, and sequenced (MWG Biotech, Ebergsberg, Germany) to exclude errors in the sequence.

Cell culture transfections and reporter assays

COS7 cell lines were maintained in exponential growth in DMEM without phenol red (Life Technologies, Inc., Invitrogen) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and 1% L-glutamine at 37 C in a 5% CO2 atmosphere. In five independent experiments performed on five different days, subconfluent COS7 cells were cotransfected in triplicate with either wild-type, variant, or control plasmids and assayed for their ability to promote transcription of the HNF1A-pGL3 reporter gene constructs. A pRL-CMV vector containing the renilla luciferase gene (Promega) was used as an internal control for the transfection efficiency. Transfections were mediated using Lipofectamine2000 (Invitrogen), and transfected cells were lysed 24 h after transfection. A dual-luciferase reporter assay system (Promega) was used to measure the ability of HNF-4{alpha} to transactivate the HNF1A-pGL3 reporter.

Western blot

Using Western blotting technique, we examined the impact of the variants on the expression of HNF-4{alpha} in COS7 cells by following a NuPAGE standard protocol. Immunoblotting was performed on lysates from COS7 cells that were transfected with 0.5 µg of a HNF4A construct (either wild-type or variant) or a standard pcDNA3 vector (basal) along with 1.0 µg reporter and 0.25 ng pRL-CMV plasmid. The estimated amount of sample (equal to 10.5 µg total protein) was loaded and electrophoretically separated on a 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane by electroblotting. Membranes were preincubated in PBS containing 0.1% Tween 20, 5% nonfat dry milk, and 0.5% BSA. Subsequently, they were incubated with a primary antibody against human HNF-4{alpha} [HNF-4{alpha}(C19); Santa Cruz, Biotechnology Inc., Santa Cruz, CA] or against ß-tubulin (horseradish peroxidase-linked anti-ß-tubulin; Santa Cruz) and a secondary antibody (antigoat IgG, horseradish peroxidase-linked; Santa Cruz). Membranes were washed in Tris-buffered saline/Tween 20 buffer, and proteins were visualized by 20 ml LumiGlo solution using a luminescent image analyzer (LAS-3000; Fujifilm, Vedbæk, Denmark) for 180 sec according to the manufacturer’s specifications.

Data analysis

The insulinogenic index was calculated as fasting serum insulin (pmol/liter) subtracted from 30-min post-OGTT serum insulin (pmol/liter) and divided by 30-min post-OGTT plasma glucose (mmol/liter). We applied a dominant model in all analyses. Differences in genotype distribution among T2D patients and control subjects were analyzed applying a Fisher’s exact test. All genotype distributions were tested for Hardy-Weinberg equilibrium using likelihood ratio tests. Differences in continuous variables were tested using a general linear model for ANOVA with adjustments for age, sex, and body mass index (BMI). All residuals were tested for normal distribution, and variables were ln-transformed if appropriate. Mean values of normalized luciferase activity (firefly luciferase activity divided by renilla luciferase activity) of the different constructs were compared using a Student’s t test (two-tail, paired). Statistical Package for Social Science (SPSS) for Windows version 12, the Web-AssoTest program (available at: http://www.ekstroem.com), or GraphPad Prism version 4 for Windows (GraphPad Software, San Diego, CA) was used for statistical analysis. A two sided P value < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Epidemiological studies of the Thr130Ile and the Val255Met polymorphisms

The results from the case-control studies of the two polymorphisms are shown in Table 2Go. The Ile allele of the Thr130Ile polymorphism was associated with a significantly increased risk of T2D [allelic frequency (95% confidence interval), 3.4% (3.1–3.9) vs. 4.2% (3.4–4.9); odds ratio, 1.26 (1.01–1.57); P = 0.04]. Among the T2D patients, carriers of the Thr130Ile polymorphism displayed a significantly higher BMI compared with noncarriers (29.7 ± 5.3 vs. 28.1 ± 4.5 kg/m2; P = 0.007), whereas no significant differences could be detected in age of diabetes onset; fasting levels of plasma glucose, serum C-peptide, serum insulin, serum triglycerides, serum cholesterol, or serum HDL cholesterol; or mode of treatment as compared with noncarriers (data not shown). In genotype-quantitative trait analyses of 4391 normal glucose-tolerant subjects, carriers of the Thr130Ile polymorphism showed significantly decreased fasting levels of serum cholesterol as compared with noncarriers (P = 0.002) and decreased fasting serum HDL cholesterol level (P = 0.03), whereas their fasting and postprandial plasma glucose, serum insulin, and serum C-peptide levels were comparable to the wild-type carriers (Table 3Go and data not shown).


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  		TABLE 2. Genotype distributions and allele frequencies for the Thr130Ile and Val255Met polymorphisms of HNF4A

 

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  		TABLE 3. Clinical and biochemical characteristics of 4391 and 4458 normal glucose-tolerant Danish white participants from the Inter99 cohort stratified according to the Thr130Ile and the Val255Met polymorphisms of HNF4A, respectively

 
The Val255Met polymorphism showed no evidence of association with T2D (Table 2Go), and no significant differences were identified in diabetes-related phenotypes among T2D patients (data not shown). However, heterozygous glucose-tolerant carriers of the Val255Met polymorphism had significantly decreased fasting serum levels of C-peptide and triglycerides as compared with glucose-tolerant noncarriers, whereas no other significant differences were identified in fasting or post-OGTT diabetes-related phenotypes (Table 3Go and data not shown).

Functional properties of Thr130Ile and the Val255Met polymorphisms

To estimate the amount of construct to be used for the transactivation studies, a titration experiment with various amounts of wild-type construct transfected into COS7 cells was performed in duplicate (data not shown). The lowest concentration of construct (0.5 µg) on the linear part of the titration curve was selected for additional experiments. Combining data from five independent experiments in triplicate, we observed a 10-fold stimulation for the wild-type HNF4A construct compared with the basal transactivation level (P < 0.001) (Fig. 1Go). The Thr130Ile and Val255Met variants showed a minor but significant decrease in activity compared with wild-type (76%, P = 0.04, and 73%, P = 0.02, respectively) (Fig. 1Go). To estimate the impact of the variants on the protein expression of HNF-4{alpha}, Western blotting was performed (Fig. 2Go). No expression of HNF-4{alpha} was observed in cells transfected with control plasmids, whereas a similar level of expression was monitored in cells transfected with the three different constructs (Fig. 2Go).



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  		FIG. 1. Combined study of five independent experiments done in COS7 cells in triplicate showing the activity levels of the HNF4A variants at a constant concentration of 0.5 µg construct along with 1.0 µg reporter construct harboring the minimal promoter of HNF1A and 0.25 ng pRL-CMV plasmid. The bars represent mean values ± SE of data transformed into fold activity with the basal activity set to 1.0. *, P < 0.05; **, P < 0.001 compared with wild-type activity.

 


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  		FIG. 2. Western blot analysis performed on lysate from COS7 cells transfected with 0.5 µg HNF4A construct (either wild-type or variant) or standard pcDNA3 vector (basal) along with 1.0 µg reporter construct harboring the minimal promoter of HNF1A and 0.25 ng pRL-CMV plasmid. A total of 10.5 µg protein was loaded into each lane and monitored using the antibody HNF-4{alpha}(C19) against human HNF-4{alpha} and using an antibody against ß-tubulin as a control.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study of Danish whites, we found that the Thr130Ile polymorphism of the HNF4A gene associated with T2D and the rare Val255Met variant associated with a decreased fasting serum C-peptide level in glucose-tolerant subjects. Both variants had decreased transcriptional activity as evaluated in an in vitro assay in COS7 cells.

The Thr130Ile variant is located in the D domain, which is involved in DNA binding (35, 36). The lower activity of the Thr130Ile variant compared with the wild-type is in accordance with a recent functional study of the same variant by Zhu et al. (37) who in HepG2 cells found a similar decrease in the transcriptional activation of the HNF1A gene. However, this impact of the polymorphism was shown only in HepG2 cells and not in HeLa or MIN6 cells (37), illustrating that a functional impact of some variants may be seen only in specific cellular contexts. In line with the results from the functional studies, we have in the present study demonstrated a significant association of the Thr130Ile with T2D with an odds ratio of 1.26. This finding is consistent with two Japanese case-control studies that together enrolled more than 500 T2D patients and 500 control subjects (37, 38). Interestingly, this odds ratio for association to T2D is similar to what has been found for the E23K variant in the ß-cell-expressed Kir6.2 gene (39). Furthermore, the effect resembles what has been found for the recently identified at-risk genotypes in other T2D susceptibility genes, Pro12Ala in the PPAR{gamma} gene (40, 41), Gly482Ser of the PGC-1{alpha} gene (42), and genetic variation in the gene encoding calpain-10 (43, 44).

The Val255Met variant showed a significant decrease in the transactivation level compared with the wild-type. This finding supports a previous study investigating the functionality of the Val255Met variant in HepG2 cells, showing a decreased functionality at a low expression vector input (0.02 µg) but not at saturation (0.3 µg) (45). The different amounts of vector used may explain the opposite finding obtained by Navas et al. (46) who failed to show any significant differences in three different cell lines. Even though not significant, the result of Navas et al. (46) showed the same tendency toward a decreased activity of the Val255Met variant compared with the wild-type.

We failed to show an association of the Val255Met polymorphism with T2D. However, in a genotype-quantitative trait interaction study, the Val255Met polymorphism was significantly associated with a decrease in fasting serum levels of C-peptide and triglycerides among 4458 glucose-tolerant subjects. The Val255Met polymorphism is positioned in one of the {alpha}-helices that are predicted to constitute the dimerization domain (47). The variant may therefore be a potential cause of reduced dimer formation and therefore influence other aspects of glucose and lipid metabolism compared with the Thr130Ile polymorphism, which is known to affect DNA binding. Furthermore, differences in frequency of these polymorphisms among the investigated population and especially the relative scarcity of the Val255Met polymorphism (Met allele frequency < 0.1%) make it hard to evaluate the impact of the variant on T2D risk. Simulation studies shows that we need more than 10,000 cases and 10,000 control subjects to have 80% power to show association to T2D with a relative risk of 1.5 given the low allele frequency. Family studies may give further insight into the T2D risk associated with a rare gene variant. In the present study, no family members were, however, available for examination. An altered HNF-4{alpha} expression or function may change regulation of important genes in the genetic transcriptional network in the ß-cells (12). This is consistent with an impact of the Val255Met leading to a significantly lower fasting serum C-peptide level.

In mice, hnf-4a is necessary for the differentiation of hepatocytes in the developing liver (48), and it holds a key role in the maintenance of the hepatic phenotype in mature hepatocytes (49). The effect of HNF-4{alpha} on numerous genes in the mature liver may include genes regulating insulin degradation. This may explain why carriers of the Val255Met variant had normal circulating serum insulin levels but decreased levels of fasting serum C-peptide. Furthermore, the serum concentrations of HDL cholesterol were decreased in the tissue-specific hepatic hnf-4{alpha} knockout mouse (49), and recent published crystallographic data show that fatty acids are endogenous ligands for HNF-4{alpha} (50). Conflicting data related to lipid homeostasis exist for both diabetic and nondiabetic carriers of MODY1 mutations. Subjects carrying the Q268X and K99fsdelAA mutations of HNF4A had lower fasting serum triglycerides and cholesterol levels compared with noncarriers (20, 51), whereas carriers of other mutations (R154X, R127W, V199I, and Phe75fsdelT) had comparable levels of these phenotypes among both diabetic and nondiabetic subjects (17, 21, 52).

Recently, identified SNPs in close proximity of the P2 promoter of the HNF4A gene were shown to associate with T2D in Finnish (31) and Ashkenazi Jewish (30) populations. In addition, further analysis of quantitative traits among unaffected offspring of Finnish T2D patients revealed an association of the at-risk allele of rs2144908 with decreased ß-cell function, but normal insulin sensitivity and normal lipid concentrations (31). This may be a result of the role of the P2 promoter in driving the HNF4A expression primarily in ß-cells (7, 8). These studies suggest that the genotype and phenotype correlation is partly explained by the site or the type of the mutation in the HNF4A gene. The exact mechanisms for the altered lipid levels in knockout mice and carriers of the MODY1 mutations are unknown. However, most HNF4A mutations identified in MODY1 patients have in vitro shown to be severe with an up to 100% decrease in transactivation activity compared with the wild-type (46 and our unpublished data). Therefore, the present findings of decreased fasting concentrations of serum cholesterol and triglycerides among carriers of the Thr130Ile and the Val255Met variants, respectively, add to the suggestion that both variants are functional.

The present study found an association of the Thr130Ile with T2D, but even with the large number of examined cases and control subjects, our study is statistically underpowered to detect an association of the Val255met variant with T2D because of the low allele frequency of the examined variant. Therefore, other large-scale epidemiological studies are needed to establish the impact of this variant.

In conclusion, the Thr130Ile and the Val255Met polymorphisms decrease the transcriptional activity of HNF4A in COS7 cells and are associated with decreased ß-cell function and altered serum lipid metabolism. The Thr130Ile polymorphism increases the risk of T2D.


   Acknowledgments
 
We thank Annemette Forman, Lene Aabo, Inge-Lise Wantzin, and Marianne Stendal for technical assistance and Grete Lademann for secretarial support.


   Footnotes
 
This work was supported by grants from the Danish Diabetes Association, the Danish Medical Research Council, The Danish Heart Foundation, and the Velux Foundation.

First Published Online February 22, 2005

Abbreviations: BMI, Body mass index; Hb, hemoglobin; HNF-4{alpha}, hepatocyte nuclear factor-4{alpha}; MODY, maturity-onset diabetes of the young; OGTT, oral glucose tolerance test; SNP, single-nucleotide polymorphism; T2D, type 2 diabetes.

Received November 2, 2004.

Accepted February 16, 2005.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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J. Ek, S. P. Hansen, M. Lajer, C. Nicot, T. W. Boesgaard, S. Pruhova, A. Johansen, A. Albrechtsen, K. Yderstraede, J. Lauenborg, et al.
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