Akihiro Inazu

Increased High-Density Lipoprotein Levels Caused by a Common Cholesteryl-Ester Transfer Protein Gene Mutation

BACKGROUND AND METHODS The plasma cholesteryl-ester transfer protein (CETP) catalyzes the transfer of cholesteryl esters from high-density lipoprotein (HDL) to other lipoproteins. We recently described a Japanese family with increased HDL levels and CETP deficiency due to a splicing defect of the CETP gene. To assess the frequency and phenotype of this condition, we screened 11 additional families with high HDL levels by means of a radioimmunoassay for CETP and DNA analysis. RESULTS We found the same CETP gene mutation in four families from three different regions of Japan. Analysis of restriction-fragment-length polymorphisms of the mutant CETP allele showed that all probands were homozygous for the identical haplotype. Family members homozygous for CETP deficiency (n = 10) had moderate hypercholesterolemia (mean total cholesterol level [+/- SD], 7.01 +/- 0.83 mmol per liter), markedly increased levels of HDL cholesterol (4.24 +/- 1.01 mmol per liter) and apolipoprotein A-I, and decreased levels of low-density lipoprotein cholesterol (1.99 +/- 0.80 mmol per liter) and apolipoprotein B. Members heterozygous for the deficiency (n = 20), whose CETP levels were in the lower part of the normal range, had moderately increased levels of HDL cholesterol and apolipoprotein A-I and an increased ratio of HDL subclass 2 to HDL subclass 3, as compared with unaffected family members (1.5 +/- 0.8 vs. 0.7 +/- 0.4). CETP deficiency was not found in six unrelated subjects with elevated HDL cholesterol levels who were from different parts of the United States. CONCLUSIONS CETP deficiency appears to be a frequent cause of increased HDL levels in the population of Japan, possibly because of a founder effect. The results that we observed in heterozygotes suggest that CETP normally plays a part in the regulation of levels of HDL subclass 2. There was no evidence of premature atherosclerosis in the families with CETP deficiency. In fact, the lipoprotein profile of persons with CETP deficiency is potentially antiatherogenic and may be associated with an increased life span.

Molecular basis of lipid transfer protein deficiency in a family with increased high-density lipoproteins.

PLASMA high density lipoproteins (HDL) are a negative risk factor for atherosclerosis. Increased HDL is sometimes clustered in families, but a genetic basis has never been clearly documented1. The plasma cholesteryl ester transfer protein (CETP) catalyses the transfer of cholesteryl ester from HDL to other lipoproteins and therefore might influence HDL levels2. Using monoclonal antibodies, we show that CETP is absent in two Japanese siblings who have markedly increased and enlarged HDL. Furthermore, they are homozygous for a point mutation in the 5′-splice donor site of intron 14 of the gene for CETP, a change that is incompatible with normal splicing of pre-messenger RNA3. The results indicate that the family has an inherited deficiency of CETP due to a gene splicing defect, and illustrate the key role that CETP has in human HDL metabolism.

Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

Circulating matrix metalloproteinases and their inhibitors in premature coronary atherosclerosis

Abstract To investigate the clinical significance of circulating matrix metalloproteinases (MMPs) and their tissue inhibitos (TIMPs) in patients with premature coronary atheroscrelosis, we studied 53 consecutive male patients with angiographically defined premature (<65 years) and stable coronary artery disease. Plasma levels of MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were determined in peripheral blood by a sandwich enzyme immunoassay, and the results were compared with those from 133 age-matched control males. There were significant differences in all the MMPs and TIMPs (p<0.001) between patients and controls. In the patient group, the levels of MMP-9 (mean±SD (ng/ml) 27.2±15.2/21.8±15.2) and TIMP-1 (130.4±55.7/94.5±26.3) were significantly higher, and the levels of MMP-2 (632.5±191.6/727.6±171.4), MMP-3 (53.1±31.2/79.6± 29.9), and TIMP-2 (24.7±15.2/35.4±16.4) were significantly lower than those of controls. We found significant positive correlation between plasma MMP-9 levels and low-density lipoprotein (LDL)-cholesterol levels (Rs=0.168, p=0.022), and significant negative correlation between plasma MMP-9 levels and high-density lipoprotein (HDL)-cholesterol levels (Rs=−0.164, p=0.026) by Spearman rank correlation test. In contrast, plasma MMP-2 (Rs=0.181, p=0.014) and MMP-3 (Rs=0.260, p=0.0004) levels were positively correlated with HDL-cholesterol levels. TIMP-2 levels were negatively correlated with total cholesterol (Rs=−0.197, p=0.007) and LDL-cholesterol (Rs=−0.168, p=0.022) levels. These results suggest that the circulating levels of MMPs and TIMPs are altered in patients with premature coronary atherosclerosis and that plasma lipoprotein cholesterol levels correlate with these, possibly as a result of the lipoprotein-vessel wall interactions.

Association of Estrogen Receptor-α Gene Polymorphisms With Coronary Artery Disease in Patients With Familial Hypercholesterolemia

To investigate the association of estrogen receptor (ER)-α gene polymorphisms with coronary artery disease (CAD), we studied 197 men and 98 postmenopausal women with heterozygous familial hypercholesterolemia. We examined the known polymorphisms, including Pvu II, Xba I, TA repeat, and CA repeat, and identified 6 novel polymorphisms in the ER-α gene. The distributions of −1989T/G (a novel polymorphism in promoter B) and Xba I in intron 1 were associated with CAD in postmenopausal women and in men, with a higher frequency of the G/G genotype ( P =0.03) or X1/X1 genotype ( P =0.02) in the CAD group. The frequency of alleles of TA repeats >17 was found to be significantly higher in postmenopausal women with CAD than in those without CAD ( P =0.04), but not in men. Logistic regression analysis with all coronary risk factors as covariates showed that the G/G genotype was a higher risk for CAD (odds ratio 4.5, 95% CI 1.0 to 19.5; P =0.04) but that X1/X1 was not. We conclude that −1989T/G or its linked polymorphisms in the ER-α gene may confer risk for CAD and that the G/G genotype may be an independent predictor for CAD in patients with familial hypercholesterolemia.

Serum lipoprotein lipid concentration and composition in homozygous and heterozygous patients with cholesteryl ester transfer protein deficiency

Six homozygous, 10 heterozygous and 8 unaffected subjects in a CETP deficient family confirmed by CETP gene analysis were studied to characterize serum lipoproteins separated by ultracentrifugation, and to examine the relations between CETP levels and lipoprotein lipid concentration and composition. The serum CETP levels were measured by radioimmunoassay using 125I-labeled monoclonal antibodies (TP2). The serum CETP levels in the homozygotes were undetectable and those in the heterozygotes were significantly lower than those in the unaffected subjects (1.5 +/- 0.1 vs. 2.2 +/- 0.5 microgram/ml, P less than 0.01). In the HDL fraction, esterified cholesterol (EC) levels in the homozygotes were significantly increased (P less than 0.01), and those in the heterozygotes were slightly increased (n.s.), in comparison with those in the unaffected and the normolipidemic controls. The EC levels in the IDL fractions were lower in the homozygotes than in the normolipidemic controls. The EC/triglyceride (TG) molar ratios in IDL, the fraction obtained from the homo- and heterozygotes, were lower than those from the unaffected subjects (P less than 0.01 and less than 0.01, respectively), and the EC/TG ratios in the HDL fraction obtained from the homo- and heterozygotes were higher than those from the unaffected subjects (P less than 0.01 and n.s., respectively). Linear regression analysis showed that positive correlates of the serum CETP levels in all subjects were: IDL-EC (r = 0.463), HDL-TG (r = 0.603) and VLDL- and IDL-EC/TG ratio (r = 0.698 and 0.843).(ABSTRACT TRUNCATED AT 250 WORDS)

Postprandial lipoprotein metabolism: VLDL vs chylomicrons.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

Long-term treatment with pitavastatin (NK-104), a new HMG-CoA reductase inhibitor, of patients with heterozygous familial hypercholesterolemia.

The clinical efficacy and safety of pitavastatin (NK-104), a novel HMG-CoA reductase inhibitor, during long-term treatment, were examined in 25 patients (male/female=11/14, mean age=53+/-13 (mean+/-SD) years) with heterozygous familial hypercholesterolemia (FH). After a period on placebo of >4 weeks, 2 mg/day of pitavastatin was administered for 8 weeks, and the dose was increased to 4 mg/day for up to 104 weeks. Total cholesterol (TC) decreased by 31% from the initial value of 340+/-57 to 237+/-40 mg/dl (P<0.0001) at week 8. During treatment with the higher dose, TC decreased even further to 212+/-35 mg/dl at week 12; it decreased by 37% from the initial value (P<0.0001). Similarly, the baseline low-density lipoprotein (LDL)-cholesterol (LDL-C) decreased by 41% at week 8, and by 49% at week 12, from 267+/-61 mg/dl at baseline. These findings indicate a dose-dependent effect of the drug on TC and LDL-C concentrations. To examine whether the levels of circulating matrix metalloproteinases (MMPs) and their endogenous inhibitors (tissue inhibitors of metalloproteinases: TIMPs) are altered during lipid-lowering therapy, we also measured their plasma levels. The mean levels of MMP-2 and -3 were significantly increased. No significant alteration was found in MMP-9, TIMP-1 and -2 levels. As for the safety of pitavastatin, adverse reactions were observed in one case (4%) of subjective and objective symptoms. The effects of pitavastatin on TC and LDL-C were stable during long treatment of patients with heterozygous FH.

Cholesteryl ester transfer protein and atherosclerosis.

Plasma cholesteryl ester transfer protein facilitates the transfer of cholesteryl ester from HDL to apolipoprotein B-containing lipoproteins. Its significance in atherosclerosis has been debated in studies of human population genetics and transgenic mice. The current review will focus on human plasma cholesteryl ester transfer protein research, including TaqIB, I405V, and D442G polymorphisms. Plasma cholesteryl ester transfer protein has a dual effect on atherosclerosis, depending on the metabolic background. In hypercholesterolaemia or combined hyperlipidaemia, plasma cholesteryl ester transfer protein may be pro-atherogenic and could be a therapeutic target.

Impacts of Visceral Adipose Tissue and Subcutaneous Adipose Tissue on Metabolic Risk Factors in Middle‐aged Japanese

Regional fat distribution rather than overall fat volume has been considered to be important to understanding the link between obesity and metabolic disorders. We aimed to evaluate the independent associations of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with metabolic risk factors in apparently healthy middle‐aged Japanese. Participants were 1,119 men and 854 women aged 38–60 years who were not taking medications for diabetes, hypertension, or dyslipidemia. VAT and SAT were measured by use of computed tomography (CT) scanning. VAT and SAT were significantly and positively correlated with each other in men (r = 0.531, P < 0.001) and women (r = 0.589, P < 0.001). In multiple regression analyses, either measure of abdominal adiposity (VAT or SAT) was positively associated with blood pressure, fasting plasma glucose, and log triglyceride (P < 0.001) and inversely with high‐density lipoprotein (HDL)‐cholesterol (P < 0.001). When VAT and SAT were simultaneously included in the model, the association of VAT with triglycerides was maintained (P < 0.001) but that of SAT was lost. The same was true for HDL‐cholesterol in women. For fasting plasma glucose, the association with VAT was strong (P < 0.001) and the borderline association with SAT was maintained (P = 0.060 in men and P = 0.020 in women). Both VAT and SAT were independently associated with blood pressure (P < 0.001). Further adjustment for anthropometric indices resulted in the independent association only with VAT for all risk factors. In conclusion, impacts of VAT and SAT differed among risk factors. VAT showed dominant impacts on triglyceride concentrations in both genders and on HDL‐cholesterol in women, while SAT also had an independent association with blood pressure.