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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1039-1044.)
© 1997 American Heart Association, Inc.

Articles

Codon 54 Polymorphism of the Human Intestinal Fatty Acid Binding Protein 2 Gene Is Associated With Dyslipidemias But Not With Insulin Resistance in Patients With Familial Combined Hyperlipidemia

Jussi Pihlajamäki; Johanna Rissanen; Sami Heikkinen; Leena Karjalainen; ; Markku Laakso

From the Department of Medicine, University of Kuopio (Finland).

Correspondence to Markku Laakso, MD, Department of Medicine, University of Kuopio, 70210 Kuopio, Finland. E-mail markku.laakso{at}uku.fi

	Abstract

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Abstract Familial combined hyperlipidemia (FCHL) is associated with variable expression of dyslipidemias and insulin resistance. In nondiabetic Pima Indians an A to G substitution in codon 54 of the fatty acid binding protein 2 (FABP2) gene has been shown to be associated with insulin resistance. We screened the entire coding region of this gene by single-strand conformation polymorphism analysis in 24 probands (17 men and 7 women; age, 63.0±7.4 years [mean±SD]; body mass index [BMI], 27.7±4.2 kg/m²) who had FCHL and in 40 healthy men from a random population sample of 82 men (age, 54.0±5.0 years; BMI, 26.3±3.2 kg/m²). Insulin resistance was assessed with the euglycemic clamp in 58 subjects from FCHL families (14 probands with FCHL and 44 first-degree relatives of probands: 38 men and 20 women; age, 51.5±12.6 years; BMI, 25.5±3.9 kg/m²). We found three nucleotide substitutions in the FABP2 gene: GCT to ACT (AlaThr) in codon 54, GTA to GTG in codon 118, and GCGCA to GCACA in the 3'-noncoding region. Frequencies of these variants did not differ between the patients and control subjects. The Ala to Thr substitution in codon 54 was associated with a high lipid oxidation rate (P=.011 after adjustment for sex and family relationship), high HDL triglycerides (P=.042), and high LDL triglycerides (P=.013) but not with insulin resistance in subjects from FCHL families. The FABP2 gene is unlikely to be a major gene for FCHL, but it might affect lipid metabolism in subjects with FCHL.

Key Words: fatty acid binding protein 2 • familial combined hyperlipidemia • insulin resistance

	Introduction

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Familial combined hyperlipidemia is characterized by a variable expression of hypercholesterolemia or hypertriglyceridemia. The prevalence of FCHL is estimated to be 1% to 2% in Western populations and may cause at least 10% of premature coronary artery disease.^{1 2} FCHL has frequently been associated with an increased rate of hepatic secretion of apoB and VLDL^{4 5 6} as well as with small dense LDL.⁷ Insulin resistance, which is associated with low HDL cholesterol,⁸ type IIB hyperlipidemia,⁹ and FCHL,¹⁰ is likely to contribute to the risk of atherosclerotic complications in this disease.¹¹

The intestinal FABP2 is an intracellular protein expressed only in the columnar absorptive epithelial cells of the small intestinal villus. The protein contains a single ligand binding site that has a high affinity for saturated and unsaturated fatty acids and has a role in the absorption and intracellular transport of long-chain fatty acids.¹² In nondiabetic Pima Indians the codon 54 (AlaThr) polymorphism was associated with a high fat oxidation rate and insulin resistance.¹³

Because the genetic basis of insulin resistance in FCHL is unknown, we screened the entire coding region of the FABP2 gene by SSCP analysis in 24 patients with FCHL and in 40 healthy men. Furthermore, we studied the association of the codon 54 polymorphism of this gene with insulin resistance and lipid and lipoprotein levels in 58 family members of patients with FCHL.

	Methods

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All subjects participating in this study were Finnish. The Finnish population is genetically quite homogeneous, descending mainly from a small number of founders of Baltic and German origin.¹⁴

Initial Screening
Twenty-four probands from families with FCHL and 40 control subjects randomly drawn from a population of 82 healthy men¹⁵ were screened for the FABP2 gene variants by SSCP analysis. The frequency of amino acid polymorphism in codon 54 of the FABP2 gene was also verified in 24 probands and in all 82 control subjects by restriction fragment length polymorphism analysis.

A total of 24 families with FCHL were included in the study. The probands were either male survivors (n=17) of myocardial infarction at a young age (<55 years) who were first studied in 1978 to 1980 and restudied in 1992 to 1994 or were their first-degree relatives fulfilling the criteria for FCHL (n=7, all women) if the male survivor of myocardial infarction had died before 1992 and could therefore not be restudied in the second examination. The criteria for FCHL were as follows: total cholesterol 7.7 mmol/L and/or total triglycerides 2.2 mmol/L in women and 2.4 mmol/L in men in at least one of the visits. These lipid criteria were based on lipid levels of 250 control subjects from a random population sample who participated in the same study as control families. The cutoff points were 80th percentile for cholesterol and 90th percentile for triglycerides. The 80th percentile for cholesterol was used because of the high cholesterol level among Finns living in eastern Finland. To meet the criteria for FCHL, all probands who were included had to have at least two affected first-degree relatives.

Control subjects (age, 54.0±5.0 years; n=82) were healthy unrelated men from our previous population-based study.¹⁵ They had a normal glucose tolerance level according to the World Health Organization criteria,¹⁶ and they did not have hypertension or symptoms or signs of coronary heart disease. All probands and control subjects had normal liver, kidney, and thyroid function tests, and none had a history of excessive alcohol intake. Because allele frequency for an autosomal polymorphism should be independent of sex, these healthy male control subjects from a random population sample could be used in estimating the allele frequencies of the variants in the FABP2 gene in the Finnish population.

Additional Screening
To study the association of the previously reported Ala to Thr amino acid substitution in codon 54 of the FABP2 gene with insulin resistance, 14 probands and 44 first-degree relatives underwent the hyperinsulinemic euglycemic clamp study to determine their insulin sensitivity.

Table 1 shows the clinical characteristics of patients with FCHL and their first-degree relatives.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Patients With FCHL and First-Degree Relatives of Patients With FCHL

Metabolic Studies
On the first day all subjects underwent an oral glucose tolerance test (75 g of glucose) after a 12-hour fast. Subjects were admitted to the metabolic ward for 1 day on a separate occasion for the euglycemic clamp study.

The degree of insulin resistance was evaluated with the euglycemic clamp technique¹⁷ after a 12-hour fast, as previously described.¹⁵ After blood was drawn for baseline measurement, a priming dose of insulin (Actrapid 100 IU/mL, Novo Nordisk) was administered during the initial 10 minutes to raise insulin concentration quickly to the desired level, where it was maintained by a continuous insulin infusion of 480 pmol (80 mU)/m² per minute. Hepatic glucose is completely suppressed. under these study conditions.^{18 19} Blood glucose was clamped at 5.0 mmol/L for the next 180 minutes by the infusion of 20% glucose at varying rates according to blood glucose measurements performed at 5-minute intervals. The mean value for the last hour was used to calculate the rates of whole body glucose uptake.

Indirect calorimetry was performed with a computerized flow-through canopy gas analyzer system (Deltatrac, Datex), as previously described.^{20 21} Gas exchange was measured for 30 minutes after a 12-hour fast and during the last 30 minutes of the euglycemic clamp procedure. The first 10 minutes of each measurement were discarded, and the mean value of the last 20 minutes was used in calculations. Protein, glucose, and lipid oxidation rates as well as energy expenditure were calculated according to Ferrannini.²² The rate of nonoxidative glucose disposal during the euglycemic clamp procedure was estimated by subtracting the carbohydrate oxidation rate (as determined by indirect calorimetry during the last 20 minutes of the euglycemic clamp procedure) from the glucose infusion rate.

Informed consent was obtained from all subjects after the purpose and potential risks of the study were explained to them. The protocol was approved by the Ethics Committee of the University of Kuopio and was in accordance with the Helsinki Declaration.

Analytical Methods
Plasma glucose levels in the fasting state as well as blood glucose and plasma lactate levels during the euglycemic clamp procedure were measured by the glucose oxidase method (2300 Stat Plus, Yellow Springs Instrument Co Inc). For the determination of plasma insulin, blood was collected in EDTA-containing tubes, and after centrifugation the plasma was stored at -20°C until the analysis. Plasma insulin concentration was determined by a commercial double-antibody solid-phase radioimmunoassay (Phadeseph Insulin RIA 100, Pharmacia Diagnostics AB). Lipoprotein fractionation was performed by the use of ultracentrifugation and selective precipitation,²³ as previously described.⁸ Cholesterol and triglyceride levels from whole serum and from lipoprotein fractions were assayed by automated enzymatic methods (Boehringer-Mannheim). ApoB was determined by a commercial immunoturbidometric method (Kone Instruments) and serum FFAs by an enzymatic method (Wako Chemicals GmbH). Serum potassium was measured by flame photometry and nonprotein urinary nitrogen by an automated Kjeldahl method.²⁴

SSCP Analysis
DNA was prepared from peripheral blood leukocytes by the proteinase K–phenol-choloroform extraction method. All four exons and the intron-exon junctions of the FABP2 gene were amplified with PCR with the use of primers, as previously reported.¹³ PCR amplification was conducted in a 10-µL volume containing 50 ng genomic DNA, 5 pmol of each primer, 10 mmol/L Tris-HCl (pH 8.8), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.1% Triton X-100, 200 µmol/L dNTP, 0.25 U DNA polymerase (Dynazyme DNA polymerase, Finnzymes), and 1.0 µCi -[³²P]dCTP. PCR conditions were denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C to 55°C for 30 to 45 seconds, and extension at 72°C for 30 to 60 seconds with final extension at 72°C for 4 minutes. SSCP analysis was performed essentially according to the method of Orita et al.²⁵ The method used in our laboratory has been shown to detect all known mutations in the lipoprotein lipase gene.²⁶ For SSCP analysis, PCR products were first diluted 10- to 20-fold with 0.1% SDS and 10 mmol/L EDTA and then diluted (1:1) with loading mix (95% formamide, 20 mmol/L EDTA, 0.05% bromphenol blue, 0.05% xylene cyanole). After denaturation at 98°C for 3 minutes, samples were immediately cooled on ice, and 2 µL of each sample was loaded onto a 6% nondenaturating polyacrylamide gel (acrylamide/N,N-methylene-bis-acrylamide ratio 49:1) containing 10% glycerol. Each sample was run at two different gel temperatures: (1) at 38°C for approximately 4 hours and (2) at 29°C for approximately 5 hours. The gel was autoradiographed overnight at -70°C with intensifying screens.

Direct Sequencing
Genomic DNA from individuals with variant single-strand conformers was used as a template in the amplification reaction as described above (total volume 50 µL, containing 25 pmol of each primer and 1.25 U of Dynazyme DNA polymerase). Amplified segments were purified by electrophoresis on a 1% low-melting-point agarose gel and directly sequenced with the use of Sequenase (US Biochemicals), as previously described.²⁷

Determination of the Frequency of the GCT to ACT Nucleotide Substitution in Codon 54 of the FABP2 Gene
This substitution disrupts the sequence of the unique Hha I restriction site in exon 2. Exon 2 was amplified with PCR in a 20-µL volume containing reagents described above in SSCP analysis. We used the following primers (5' to 3'): FEX2F (forward)=CACTTCCTATGGGATTTGACT and FEX2R (reverse)=TTGGGTAGAAAAATCAAGAATG (product size, 274 bp).¹³ Amplified segments of exon 2 were digested with Hha I and separated through a 3% agarose gel (NuSieve, FMC BioProducts). PCR products lacking the Hha I restriction site migrated as 274-bp fragments, whereas PCR products containing an intact Hha I site are cleaved into 149-bp and 125-bp fragments.¹³

Statistical Analysis
All data are presented as mean±SD. Statistical analyses were performed with the SPSS/PC+ programs (SPSS Inc). The frequencies between the study groups were compared with the ² test. Continuous variables were compared with Student's two-tailed t test. In comparison of two groups, the adjustment for confounding factors was done with ANCOVA. Because of skewed distributions, insulin, VLDL cholesterol, all triglyceride fractions, and FFAs were logarithmically transformed. After logarithmic transformation, each of these variables had a normal distribution.

	Results

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The frequency of the Thr54 encoding allele of the FABP2 gene did not differ between patients with FCHL and control subjects (Table 2). In addition, we found two other nucleotide substitutions: a silent substitution of GTA to GTG in exon 4 in codon 118, and a substitution of GCGCA to GCACA in the 3' noncoding region (15 bp downstream from the coding region of exon 4). The frequencies of these variants as well as the allele frequencies for the variable lengths of ATT repeat sequence in intron 2 did not differ between patients and control subjects (Table 2).

View this table: [in this window] [in a new window]   Table 2. Frequencies of Variants in the FABP2 Gene in Control Subjects and Patients With FCHL

The subjects with genotypes of Thr54Thr or Ala54Thr were pooled in analyses concerning the effects of the codon 54 polymorphism of the FABP2 gene on insulin sensitivity and lipid metabolism because of a small number of subjects who were homozygous for the Thr54 allele (n=6). A total of 25 subjects had the genotype Ala54Ala, and 33 subjects had the Thr54 allele. Age (49.2±12.8 versus 53.9±12.2 years) and body mass index (25.8±3.1 versus 25.3±4.5 kg/m²) did not differ between these groups, but sex distribution was different (20 men and 5 women versus 18 men and 15 women; P=.043). Fasting FFA levels as well as fasting lipid oxidation rates were similar in subjects with the Ala54Ala genotype and with the Thr54 allele (Table 3). However, when the subjects with the genotypes of Thr54Thr, Ala54Thr, and Ala54Ala were compared with respect to fasting lipid oxidation rate after adjustment for sex, the groups differed significantly (1.14±0.21 versus 0.86±0.25 versus 0.81±0.24 mg/kg per minute; P=.008). This difference remained statistically significant even after adjustment for family relationship (P=.011).

View this table: [in this window] [in a new window]   Table 3. Results of the Euglycemic Clamp Study in Patients With FCHL and in First-Degree Relatives of Patients With FCHL According to Codon 54 Polymorphism of the FABP2 Gene

Insulin resistance determined as the rate of whole body glucose uptake during the last hour of the euglycemic clamp procedure was not different between the groups (53.2±13.4 versus 54.7±15.7 µmol/kg per minute). Similarly, the rates of glucose oxidation and nonoxidation during the last 30 minutes of the clamp study as well as the suppression of FFAs, the rate of lipid oxidation, and energy expenditure during the last 30 minutes of the clamp study did not differ between the groups.

When the results presented in Table 3 with the exception of lipid oxidation rate were compared among the three genotypes (Thr54Thr, Ala54Thr, and Ala54Ala), no statistically significant differences were found.

The subjects with the Thr54 allele had higher sex-adjusted HDL triglycerides (P=.040) and LDL triglycerides (P=.013) than those who had the Ala54Ala genotype (Table 4). When the results were adjusted, in addition to family relationship the differences in HDL triglycerides (P=.042) and LDL triglycerides (P=.013) still remained statistically significant. We also analyzed these results among affected and nonaffected family members. Family members who had FCHL according to our lipid criteria (n=26) and who had the Thr54 allele (n=17) had 20% higher LDL cholesterol (5.65±1.09 versus 4.71±0.98 mmol/L; P=.053), 27% higher HDL triglyceride (0.28±0.10 versus 0.22±0.04 mmol/L; P=.148), and 34% higher LDL triglyceride (0.58±0.24 versus 0.38±0.16 mmol/L; P=.068) levels than subjects with FCHL and the Ala54Ala genotype (n=9). These trends were not found in nonaffected family members (n=32; 16 subjects had the Thr54 allele and 16 had the Ala54Ala genotype). Compared with the subjects with the Ala54Ala genotype, subjects with the Thr54 allele had 2.7% lower LDL cholesterol (P=.719), 10% higher HDL triglyceride (P=.318), and 21% higher LDL triglyceride (P=.252) levels. No differences in fasting glucose and insulin levels were found between the groups.

View this table: [in this window] [in a new window]   Table 4. Plasma Glucose, Plasma Insulin, and Serum Lipids and Lipoproteins in Patients With FCHL and in First-Degree Relatives of Patients With FCHL According to Codon 54 Polymorphism of the FABP2 Gene

When the levels of fasting glucose, insulin, and serum lipids and lipoproteins were compared between the three genotypes (Thr54Thr, Ala54Thr, and Ala54Ala), no statistically significant differences were found.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Insulin resistance is a characteristic feature in FCHL.^{9 10} Since genetic variation in the FABP2 gene could be the cause of insulin resistance, we screened for variants in this gene in patients with FCHL. According to our study, the FABP2 gene is not likely to be a major gene for FCHL. However, the polymorphism in codon 54 of this gene (AlaThr) may contribute to abnormalities in lipid metabolism in FCHL, although it does not have a significant effect on insulin resistance.

Complex segregation analysis has suggested a major gene effect on apoB²⁸ and on triglycerides²⁹ in FCHL. Although some uncommon variants in the lipoprotein lipase gene²⁶ could cause FCHL, no gene that could explain a major part of this disease has yet been found. We found one novel nucleotide substitution in the 3' noncoding region (AG) of the FABP2 gene in patients with FCHL. In addition, we found nucleotide substitutions in codon 54 (GCTACT) and in codon 118 (GTAGTG) described previously in Pima Indians.¹³ Since the frequency of these substitutions and ATT repeat sequence lengths in intron 2 did not differ between the patients with FCHL and control subjects, the possibility that the FAPB2 gene could play a major role in the etiology of FCHL in the Finnish population is very unlikely.

In our study insulin sensitivity was not associated with the codon 54 polymorphism of the FABP2 gene, in contrast to results in nondiabetic Pima Indians.¹³ The reason for these opposite findings remains unexplained, but the effect of the codon 54 polymorphism on insulin resistance could be population-specific. It is interesting to note that the association of the Thr54 allele and high lipid oxidation rate described in Pima Indians was verified in the present study because fasting lipid oxidation rate differed between subjects with Thr54Thr, Ala54Thr, and Ala54Ala genotypes. The subjects who were homozygous for the Thr54 allele had the highest fasting lipid oxidation rate.

The novel finding in this study was that high HDL triglyceride and high LDL triglyceride levels were found in subjects with the Thr54 allele of the FABP2 gene. Although these associations were rather weak and could therefore also be chance findings, they are theoretically interesting because the Thr54-containing protein has been associated with increased binding of fatty acids¹³ and therefore might change lipid metabolism. This theory is supported by the association between the Thr54 allele with increased lipid oxidation rate in Pima Indians¹³ and in the present study. The mechanisms by which codon 54 polymorphism could lead to increased lipid oxidation rate and dyslipidemias is unknown. Because subjects who have FCHL are prone to postprandial lipemia^{30 31} and because the associations of the Thr54 allele and dyslipidemias tended to be stronger in family members with FCHL, changes in postprandial lipid metabolism could at least in part contribute to dyslipidemias. For example, high levels of postprandial serum FFAs could lead to increased hepatic synthesis of VLDL and apoB,³² dyslipidemias, and insulin resistance.³³ However, we did not measure serum FFAs after a normal fat-containing meal.

LDL particles were richer in triglycerides in subjects with the Thr54 allele than in subjects with the Ala54Ala genotype of the FABP2 gene. This compositional change in LDL particles often indicates that LDL is small and dense.^{34 35} Because small dense LDL particles are associated with FCHL⁸ ,^{36 37} and coronary artery disease,³⁸ an atherogenic composition of LDL particles seems to be associated with the Thr54 allele in FCHL. Direct measurement of LDL particle size is needed to confirm this finding.

We conclude that the FABP2 gene is not likely to be a major gene in FCHL. However, the polymorphism in codon 54 of this gene may influence lipid oxidation rate or lipid and lipoprotein levels in subjects with FCHL.

	Selected Abbreviations and Acronyms

 

apoB = apolipoprotein B FABP2 = fatty acid binding protein 2 FCHL = familial combined hyperlipidemia FFAs = free fatty acids PCR = polymerase chain reaction SSCP = single-strand conformation polymorphism

	Acknowledgments

 
This study was supported by a grant from the Medical Research Council of the Academy of Finland.

Received May 2, 1996; accepted October 14, 1996.

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