Norimichi Nakajima

Molecular mechanisms, lipoprotein abnormalities and atherogenicity of hyperalphalipoproteinemia.

Hyperalphalipoproteinemia (HALP) is caused by a variety of genetic and environmental factors. Among these, plasma cholesteryl ester transfer protein (CETP) deficiency is the most important and frequent cause of HALP in the Asian populations. CETP facilitates the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to apolipoprotein (apo) B-containing lipoproteins, and is a key protein in the reverse cholesterol transport system. The deficiency of CETP causes various abnormalities in the concentration, composition, and function of both HDL and low density lipoprotein (LDL). The significance of CETP in terms of atherosclerosis had been controversial. However, the in vitro evidence showed large CE-rich HDL particles in CETP deficiency are defective in cholesterol efflux. Similarly, scavenger receptor BI (SR-BI) knockout mice show a marked increase in HDL-cholesterol but accelerated atherosclerosis in atherosclerosis-susceptible mice. Recent epidemiological studies in Japanese-Americans and in Omagari area where HALP subjects with the intron 14 splicing defect of CETP gene are markedly frequent, have demonstrated an increased incidence of coronary atherosclerosis in CETP-deficient patients. Thus, CETP deficiency is a state of impaired reverse cholesterol transport which may possibly lead to the development of atherosclerosis. The current review will focus on the molecular mechanisms and atherogenicity of HALP, especially CETP deficiency.

Prevalence and phenotypic spectrum of cholesteryl ester transfer protein gene mutations in Japanese hyperalphalipoproteinemia.

A patient with cholesteryl ester transfer protein (CETP) deficiency presents with marked hyperalphalipoproteinemia (HALP). To investigate the contribution of CETP deficiency to the cause of HALP (HDL-C> or =1.94 mmol/l, 75 mg/dl), we investigated the CETP activities and the prevalence of genetic CETP mutations among 624 Japanese HALP subjects. The subjects were screened for four known genetic CETP mutations (intron 14 splicing defect (In14), exon 15 missense mutation (Ex15), intron 10 splicing defect (In10) and exon 6 nonsense mutation (Ex6)). We found the frequency of the patients with reduced CETP activity (<75% of normal controls) to be 55.5 and 64.1% in a high HDL group (1.94< or =HDL-C<2.59 mmol/l) and a marked HALP group (HDL-C> or =2.59 mmol/l, 100 mg/dl), respectively. At least one of the four mutations was identified in 65.7% of subjects with reduced CETP activities and 57.5% of subjects with marked HALP. The In14 and Ex15 mutations were very common in HALP subjects and the frequency of In10 mutation and Ex6 mutation was quite low. To investigate the impact of genetic CETP mutation on the phenotypes, we compared the plasma lipid levels and CETP activities between the subjects with two common mutations. All In14 homozygotes showed marked HALP, while marked HALP is less frequent (64.3%) in Ex15 homozygotes. HDL-C levels in Ex15 heterozygotes were significantly higher than those of In14 heterozygotes, suggesting the mutation has dominant negative effects on CETP activity in vivo. Some cases with In14 (5.7%) or Ex15 (7.2%) mutation showed low HDL-C levels. We conclude that CETP deficiency is a major cause of HALP; nevertheless CETP deficiency is not necessarily HALP.

Frequency of exon 15 missense mutation (442D:G) in cholesteryl ester transfer protein gene in hyperalphalipoproteinemic Japanese subjects

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester from high density lipoprotein (HDL) to apo B-containing lipoproteins. The hyperalphalipoproteinemia caused by CETP deficiency is fairly common in Japan and one of the most common mutations in the CETP gene is the splicing defect of the intron 14, the allelic frequency of which has been shown to be 0.0049 in the Japanese general population. Recently, we have reported a missense mutation in exon 15 of the CETP gene (442D:G), showing a dominant effect on the CETP activity and HDL-cholesterol level. In the current study, we determined the frequency of this new mutation in Japanese hyperalphalipoproteinemic (HDL-cholesterol > or = 100 mg/dl) subjects. A rapid and easy screening method for this new mutation was developed using a polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Among 117 Japanese hyperalphalipoproteinemic subjects (HDL-cholesterol; 116.7 +/- 16.5 mg/dl, mean +/- S.D.) without the intron 14 splice defect, three homozygotes (2.5%) and 34 heterozygotes (29.1%) were found to have the 442D:G mutation. The relative allelic frequency of this mutation was calculated to be 0.17. One of the homozygotes for the 442D:G mutation was the patient previously described by us as having hyperalphalipoproteinemia with corneal opacity and coronary heart disease. This was the first reported subject homozygous for the CETP deficiency who also demonstrated atherosclerotic symptoms. In homozygous subjects, CETP activity ranged from 37% to 62% of the normal value, which was consistent with the results obtained from the transient expression experiment previously reported; however, the specific activity of CETP was not as low as expected.(ABSTRACT TRUNCATED AT 250 WORDS)

Point Mutation (−69 G→A) in the Promoter Region of Cholesteryl Ester Transfer Protein Gene in Japanese Hyperalphalipoproteinemic Subjects

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) from HDL to apolipoprotein (apo) B–containing lipoproteins and plays a crucial role in reverse cholesterol transport, which is a major protective system against atherosclerosis. Genetic CETP deficiency is the most common cause of a marked hyperalphalipoproteinemia (HALP) in the Japanese, and various mutations have been identified in the coding region as well as in the exon/intron boundaries in the CETP gene. In the present study, we identified a novel mutation in the promoter region of the CETP gene. This mutation was a G-to-A substitution at the −69 nucleotide of the promoter region (−69 G→A), corresponding to the second nucleotide of the PEA3/ETS binding site (C G GAA) located upstream of the putative TATA box. Four (2.0%) of 196 unrelated subjects with a marked HALP (HDL cholesterol ≥2.59 mmol/L=100 mg/dL) were revealed to be heterozygous for the −69 G→A mutation, and the allelic frequency of the mutant was 0.0102 in the subjects with a marked HALP. The subjects with the −69 G→A mutation had low plasma CETP levels. Reporter gene assay showed that this mutation markedly reduced the transcriptional activities in HepG2 cells (8% of wild type). These results suggested that this mutation would be dominant negative. In conclusion, a novel −69 G→A mutation in the CETP gene causes the decreased transcriptional activity leading to HALP.

Impairment of Reverse Cholesterol Transport System and Atherosclerosis

High density lipoprotein (HDL) derives cholesterol from peripheral tissues, and cholesterol is esterified by lecithin:cholesterol acyltransferase (LCAT), forming cholesteryl ester (CE). HDL serves as a carrier of cholesterol from tissues to the liver for excretion into the bile. This system was designated as “ reverse cholesterol transport”, which protects against atherosclerosis. Plasma cholesteryl ester transfer protein (CETP) promotes the transfer of CE from HDL to apolipoprotein (apo) B-containing lipoproteins such as very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Since the CETP reaction theoretically reduces plasma HDL-cholesterol and increases VLDL and LDL-cholesterol, CETP has been considered to be a proatherogenic factor and its deficiency has been presumed to be an anti-atherogenic state. However, the discovery of CETP-deficient subjects and the detailed analysis of the lipoprotein metabolism in these patients revealed the important roles of CETP in the metabolism of both HDL and LDL particles and in the reverse cholesterol transport system. CETP deficiency causes a variety of abnormalities in the concentration, composition and functions of both HDL and LDL. Our in vitro study demonstrated that large CE-rich HDL particles, which accumulate in the plasma of CETP-deficient subjects, have an impaired ability for cholesterol efflux from lipid-laden macrophages. The addition of CETP into the plasma of CETP-deficient subjects induced the formation of small very high density lipoprotein (VHDL) particles enriched with protein and phospholipids. These VHDL particles had a potent antiatherogenic function when incubated with acetyl-LDL loaded macrophages. Furthermore, our recent epidemiological studies have demonstrated an increased incidence of atherosclerosis in the coronary and carotid arteries of CETP-deficient subjects. Two common mutations have been identified in CETP deficiency; one is a G-to-A mutation at the 5’ splice donor site of intron 14 and the other is a missense mutation (D442:G). We found that the G-to-A mutation at the 5’ splice donor site of intron 14 in the CETP gene is extremely frequent in Omagari area of Akita Prefecture. Epidemiological studies in this area demonstrated that CETP-deficient subjects are not accompanied by longevity and that atherosclerotic cardiovascular diseases can often develop in these patients. Therefore, a marked hyperalphalipoproteinemia due to CETP deficiency may be a proatherogenic state, suggesting the importance of analyzing the function of HDL in vivo.