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Association of I27L Polymorphism of Hepatocyte Nuclear Factor-1 Gene with High-Density Lipoprotein Cholesterol Level

Department of Geriatric Medicine (N.B., H.I., T.F., K.N., M.I.-B., K.I., T.O.), Osaka University Graduate School of Medicine, Osaka 565-0871; Department of Geriatric Medicine (J.N., M.A., M.Y., J.J.J., Z.W., T.M.), Ehime University School of Medicine, Ehime 791-0295; and Department of Ophthalmology (M.F.), Nippon Telegraph and Telephon West Osaka Hospital, Osaka 543-0042, Japan

Address all correspondence and requests for reprints to: Hiroshi Ikegami, M.D., Ph.D., Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ikegami{at}geriat.med.osaka-u.ac.jp.

	Abstract

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The serum level of high-density lipoprotein cholesterol (HDL-c), which protects against the development of atherosclerosis, is under genetic control. However, the genetic components responsible for the serum HDL-c level are yet to be determined. A recent knockout mouse study demonstrated that hepatocyte nuclear factor-1 (HNF-1) is an essential transcriptional regulator of HDL-c metabolism. In this study, the association of an HNF-1 gene polymorphism, isoleucine (Ile) 27 leucine (Leu), with lipid parameters, in particular with serum HDL-c level, was studied in 356 unrelated Japanese men. Though no significant difference was observed in total cholesterol and triglyceride levels among the three genotypes, the serum HDL-c level was significantly associated with the genotype (P < 0.01, trend test). Subjects with the Ile/Ile genotype had low serum HDL-c levels, and those with the Leu/Leu genotype had high serum HDL-c levels. These results demonstrate that the HNF-1 gene locus is associated with serum HDL-c level and suggest that the Ile27 allele is a risk marker for atherosclerosis.

	Introduction

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HIGH-DENSITY LIPOPROTEIN-cholesterol (HDL-c) protects against the development of atherosclerosis (1), and low serum HDL-c is generally accepted as a strong and independent risk factor for the development of coronary artery disease and cerebrovascular disease (1, 2, 3). Although environmental factors are known to be involved in alteration of lipid profile (4, 5), both segregation analyses and twin studies indicated that serum HDL-c level is under strong genetic influence; i.e. almost one half of the population variance of HDL-c level is explained by genetic factors (4). The genetic components responsible for its variation are, however, yet to be determined.

Apolipoproteins are key regulators of the lipid profile. Among apolipoproteins, apolipoprotein A-I (apo A-I) is particularly important for HDL-c metabolism (6, 7, 8). Hepatocyte nuclear factor (HNF)-1, a transcription factor expressed in the liver (9, 10), has been shown to modulate transcription of apo A-I (11), suggesting a role of HNF-1 in HDL-c metabolism. In fact, a recent study in HNF-1 knockout mice demonstrated that HNF-1 is essential in the regulation of HDL-c metabolism (12). The HNF-1 gene, transcription factor 1 (TCF1), is located on chromosome 12q24.2 (13), where genes responsible for diabetes-related phenotypes have been mapped in previous studies with genome-wide scanning (14, 15, 16). Therefore, TCF1 is a functional and positional candidate gene responsible for serum HDL-c level.

In the present study, we studied the association of a single-nucleotide polymorphism (SNP), isoleucine (Ile) 27 leucine (Leu) of TCF1 (17), with lipid profile in the Japanese population to clarify whether the TCF1 locus is involved in determining the serum HDL-c level.

	Subjects and Methods

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Subjects

A total of 356 unrelated Japanese men, 51.3 ± 8.7 yr old (mean ± SD), who attended annual health checkups, were studied. None of the subjects was receiving a lipid-lowering agent. Informed consent was obtained from all the subjects.

Methods

Body mass index (BMI) was calculated as body weight (kg) divided by the square of height (m). Systolic and diastolic blood pressure were measured using a mercury sphygmomanometer. Blood samples were collected after overnight fasting, and serum total cholesterol (T-chol), triglyceride, and HDL-c levels were measured with standard protocols. Low-density lipoprotein-cholesterol (LDL-c) was estimated using the formula of Friedewald et al. (18). Because serum HDL-c level differs among countries (1, 19), the subjects were divided into two groups [high-HDL group (HDL-c 1.03 mg/dl) and low-HDL group (HDL-c < 1.03 mg/dl)], according to the Japan Atherosclerosis Society Guideline (1).

Genotypes of the Ile27Leu polymorphism in TCF1 were determined by PCR-restriction fragment length polymorphism method, as reported previously (20). Briefly, genomic DNA was extracted from peripheral blood leukocytes by a standard phenolchloroform method. PCR was performed using the following oligonucleotides: (sense) 5'-GAGCCATGGTTTCTAAACTG-3' and (antisense) 5'-TCTCCAGCCAGGAGGTAG-3', and amplification was performed with initial denaturation for 3 min at 94 C, followed by 40 cycles of 1 min at 94 C, 1 min at 60 C, and 1 min at 72 C. The PCR product was digested with Mbo I at 37 C, followed by electrophoresis in 9% acrylamide gels, and stained with ethidium bromide.

Statistical analysis

Data are shown as mean ± SD, unless otherwise stated. The ² test was used for categorical variables. The phenotype difference among the genotype groups was tested by trend test (Jonckheere test). A P value less than 0.05 was regarded as significant.

	Results

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The genotype frequency of Ile/Ile, Ile/Leu, and Leu/Leu of the subjects was 24.2%, 55.3%, and 20.5%, respectively. This distribution was compatible with the Hardy-Weinberg equilibrium. The clinical and biochemical characteristics of the subjects, according to the TCF1 genotype, are shown in Table 1. No significant difference was found in age, height, body weight, BMI, and blood pressure among the genotype groups. T-chol, triglyceride, and LDL-c levels were not significantly different among the three groups with different genotypes. Serum HDL-c level, however, was significantly associated with the number of Leu27 alleles (P < 0.01, trend test); i.e. subjects with the Ile/Ile genotype had low serum HDL-c levels, those with the Ile/Leu genotype had moderate serum HDL-c levels, and those with the Leu/Leu genotype had high serum HDL-c levels. When the subjects were divided into two groups according to serum HDL-c level, the genotype distribution was significantly different (P < 0.05) (Table 2), and the frequency of the Leu allele in the low-HDL-c group was significantly lower than that in the high-HDL-c group (P < 0.01). When the subjects were divided into quintiles according to serum HDL level, the genotype distribution was significantly (P < 0.05) different between the lowest and highest groups.

View this table: [in this window] [in a new window]   Table 1. Ile27Leu genotypes of HNF-1 gene and clinical and biochemical characteristics

View this table: [in this window] [in a new window]   Table 2. Genotype and allele frequencies of Ile27Leu polymorphism of HNF-1 gene according to HDL-c status

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HDL-c level is shown to be under strong genetic control (4), but the genetic components responsible for its variation are largely unknown. The present study demonstrated a significant association of the Ile27Leu polymorphism of the HNF-1 gene, a key transcriptional regulator of HDL-c metabolism, with serum HDL-c level in healthy Japanese men. The results suggest that the TCF1 locus is involved in the genetic variation in serum HDL-c level.

Epidemiological studies have suggested a role of genetic factor(s) in atherosclerotic diseases. Candidate gene approaches have revealed some genetic components predisposing to atherosclerotic disease, but the majority of the genetic components are still unknown. Given the well-known antiatherosclerotic role of HDL-c (8) and the role of HNF-1 in transcriptional regulation of apo A-I (11), a key apolipoprotein in HDL-c metabolism, the HNF-1 gene is a strong candidate gene for atherosclerotic disease. The significant association of the Ile27Leu polymorphism with serum HDL-c level, as shown in the present study, suggests that the polymorphism may also be associated with atherosclerosis. The association of the polymorphism with the index, T-chol/HDL-c, which has previously been shown to be a powerful predictor of atherosclerosis (21, 22, 23, 24), supports this possibility. Further studies on the association of the polymorphism with atherosclerosis are necessary to address this possibility.

The Oji-Cree of northern Canada have a higher prevalence of type 2 diabetes and higher BMI than Asian populations, which makes it difficult to see the effect of genetic factors on lipid-related phenotypes. Despite this difficulty, the association of an SNP of the HNF-1 gene (G319S) with HDL-c level (25) has recently been reported in the Oji-Cree. Although the SNP associated with HDL-c in their study was different from that in our study, the finding of an association in two different ethnic groups strongly suggests that serum HDL-c level is genetically controlled by the TCF1 locus.

In this study, the Ile27 allele of the TCF1 polymorphism was also associated with decreased serum HDL-c level. The precise molecular mechanism linking the SNP with a low HDL-c level is unknown, but two explanations are plausible. First, the SNP itself may be functional and may directly influence the serum HDL-c level. This SNP results in an amino acid substitution, Ile to Leu, in the dimerization domain of HNF-1 (26, 27). Because HNF-1 is biologically active in a dimer form, either as a homodimer or as a heterodimer with HNF-1ß (28), the amino acid substitution may alter its three-dimensional structure and affect the dimer formation. Another possibility is that the Ile27Leu polymorphism is in linkage disequilibrium with another putative functional polymorphism of the HNF-1 gene, which might affect transcription activity of the HNF-1 molecule or its expression. In either case, given the lack of pharmacological agents that specifically increase serum HDL-c level, HNF-1 may be an important target molecule for the development of drugs to prevent atherosclerosis by increasing HDL-c level, if the HNF-1 gene itself is involved in the regulation of serum HDL-c level.

In the Japanese subjects in the present study, the mean HDL-c level was higher than that generally reported in a Caucasian population, which is consistent with observations reported previously (1, 29, 30). The reason for the racial difference has not been clarified, but it is possible that the difference is attributable to genetic, environmental, or cultural differences (4). The lower frequency of the Ile allele in the Japanese population (51.8%) than in a Caucasian population (63%) (17) might contribute, to some extent, to the difference between the two populations.

The association of the Ile27 allele with serum HDL-c level was dose-dependent, indicating a codominant mode of inheritance. Several mutations in the HNF-1 gene have been reported as causes of maturity-onset diabetes of the young (MODY) (17). These mutations are reported to act on diabetes-related phenotypes in a dominant fashion. The difference in the modes of inheritance (codominant vs. dominant) may be explained by the severity of the dysfunction caused by mutations; i.e. a more severe mutation (as reported for MODY) shows a dominant mode of inheritance, and a less severe mutation (as in the present study) shows a codominant mode of inheritance. A more fundamental question is why some polymorphisms of one gene cause diabetes (17), whereas another is associated with HDL-c level without affecting susceptibility to type 2 diabetes (20). This may be possibly explained by the difference in transcriptional networks between pancreatic ß-cells and hepatocytes.

In conclusion, the present study indicated that the Ile27 allele of the HNF-1 gene was inversely associated with serum HDL-c level. These data suggest that the HNF-1 gene may be involved in the regulation of serum HDL-c level and that the Ile27 allele can serve as a risk marker for atherosclerosis.

	Acknowledgments

 
We thank Miyuki Moritani for skillful technical assistance.

	Footnotes

 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

Abbreviations: apo A-I, Apolipoprotein A-I; BMI, body mass index; HDL, high-density lipoprotein; HDL-c, HDL cholesterol; HNF, hepatocyte nuclear factor; Ile, isoleucine; LDL, low-density lipoprotein; LDL-c, LDL cholesterol; Leu, leucine; MODY, maturity-onset diabetes of the young; SNP, single-nucleotide polymorphism; TCF1, transcription factor 1; T-chol, total cholesterol.

Received November 30, 2002.

Accepted February 27, 2003.

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