Cholesteryl Ester Transfer Protein Deficiency Research Articles

4.P.350 Mutational analysis of hyperalphalipoproteinemia (HALP) in the Japanese: Cholesteryl ester transfer protein deficiency contributes about 60% of marked HALP

4.P.350 Mutational analysis of hyperalphalipoproteinemia (HALP) in the Japanese: Cholesteryl ester transfer protein deficiency contributes about 60% of marked HALP

4.C.5 Cholesteryl ester transfer protein deficiency: Virtue or vice?

4.C.5 Cholesteryl ester transfer protein deficiency: Virtue or vice?

1.P.270 Human plasma CETP deficiency: A new mutation in the first Caucasian North American subject

1.P.270 Human plasma CETP deficiency: A new mutation in the first Caucasian North American subject

Clinical characteristics of double heterozygotes with familial hypercholesterolemia and cholesteryl ester transfer protein deficiency

Coronary heart disease (CHD) in familial hypercholesterolemia (FH) may be modified by genetic and/or environmental factors. We described the effect of the cholesteryl ester transfer protein (CETP) gene on CHD in heterozygous FH caused by low density lipoprotein receptor (LDL-R) gene mutation. In 288 unrelated Japanese subjects with heterozygous FH, the allele frequency of an intron 14 G(+1)-to-A mutation (Int14 A) and a missense mutation in exon 15 (Asp 442 to Gly, D442G) was 0.3 and 3.0%, respectively. HDL-C levels (1.55±0.08 mmol/l) in FH patients with heterozygous CETP deficiency were higher than those (1.19±0.08 mmol/l) in FH without CETP deficiency ( P<0.03), while LDL-C levels in FH with CETP deficiency were moderately reduced. However, two FH patients with CETP deficiency suffered myocardial infarction, and six patients had effort angina pectoris and/or coronary atherosclerosis. No difference in the score of coronary stenosis index (CSI) was found in FH with/without CETP deficiency, although CSI was inversely correlated with HDL-C levels ( P<0.05). Thus, the effect of increased HDL-C levels caused by partial deficiency of CETP is insufficient to prevent CHD in FH.

Skipping of Exon 14 and Possible Instability of Both the mRNA and the Resultant Truncated Protein Underlie a Common Cholesteryl Ester Transfer Protein Deficiency in Japan

Among the Japanese population, a G-to-A mutation at the beginning of intron 14 of the human cholesteryl ester transfer protein (CETP) gene is a frequent cause of CETP deficiency characterized by markedly increased HDL cholesterol. The resulting abnormalities responsible for null CETP deficiency were studied in detail. The CETP mRNA transcripts amplified by polymerase chain reaction from the monocyte-derived macrophages of two homozygous patients were both found to be normal except for the whole deletion of exon 14. The deletion causes a shift of reading frame and introduces a premature termination codon downstream. Examination of the macrophage RNA from heterozygotes suggested the increased instability of the abnormal mRNA in the cytoplasm, because the amount of the aberrant transcript was nearly one third that of a normal transcript in the cytoplasm, while they were equal in the nucleus. Although this indicated the synthesis of a mutant CETP that lacks about 15% at its carboxy terminus, immunoblot analysis demonstrated that the abnormal CETP was virtually absent in both the media and cell lysates of transfected COS-1 cells, which massively expressed the mutant CETP mRNA. These results elucidate the primary abnormality due to the common CETP splicing mutation to be the exon skipping of mRNA, which decreases the level of mRNA and produces a truncated protein that should be rapidly degraded intracellularly.

Quantitative and compositional changes in high density lipoprotein subclasses in patients with various genotypes of cholesteryl ester transfer protein deficiency

High density lipoprotein (HDL) with and without apolipoprotein (apo) E was quantified and characterized in subjects with three genotypes of cholesteryl ester transfer protein (CETP) deficiency: the nonsense mutation in intron 14 (10 homozygotes and 5 heterozygotes); the missense mutation in the exon 15 (3 homozygotes and 9 heterozygotes); and the Int14A/D442G in 6 compound heterozygotes. ApoE-poor and apoE-rich HDL-cholesterol levels were elevated significantly in all genotypic groups with the decrease in CETP activity, indicating that both types of HDL-cholesterol can be a substrate for CETP. However, an unchanged or only slightly increased serum apoA-II level in each genotype indicated that the HDL particles with apoA-II are relatively resistant to CETP-mediated lipid transfer. Serum apoE-rich HDL level was considerably higher in the Int14A homozygotes than in the compound heterozygotes, in spite of similar apoE-poor HDL-cholesterol levels, which may indicate that apoE-rich HDL is a better substrate for CETP than apoE-poor HDL. Although the apoE-rich and apoE-poor HDL subclasses were similar in the accumulation of cholesteryl ester and depletion of triglyceride, the accumulation of free cholesterol was unique to apoE-rich HDL, indicating inhibited cholesterol esterification on this lipoprotein. Clinical laboratories should be aware of the discrepancy in HDL-cholesterol measurements that comes from the different recoveries of apoE-rich HDL using commercial reagents. In conclusion, CETP deficiency causes considerable quantitative and compositional changes in HDL subclasses, reflecting a significant physiological role for CETP in HDL metabolism.

Genetic cholesteryl ester transfer protein deficiency is extremely frequent in the Omagari area of Japan. Marked hyperalphalipoproteinemia caused by CETP gene mutation is not associated with longevity.

Low levels of HDL cholesterol have been clearly demonstrated to be associated with an increased incidence of coronary heart disease, strongly suggesting that HDL particles have an antiatherogenic function. However, little information has been available concerning the atherogenicity of a marked hyperalphalipoproteinemia (HALP). There is no agreement about whether plasma cholesteryl ester transfer protein (CETP) deficiency is associated with an antiatherogenic state or not, although this disorder was reported to be one of the major causes of marked HALP. In the current study, we have found a unique area (Omagari City, Akita Prefecture, Japan) where CETP deficiency caused by a G-to-A mutation at the 5' splice donor site of intron 14 in the CETP gene is extremely frequent. In Omagari City, the mutation was detected more than 20 times more frequently and the prevalence of a marked HALP with plasma HDL cholesterol > or = 2.58 mmol/L (100 mg/dL) was 5 to 10 times higher than in other areas of Japan. This discovery has made it possible to perform a large population-based study concerning the atherogenicity of a marked elevation of HDL cholesterol in a genetically more homogeneous population. There was a statistically significant U-shaped relationship between HDL cholesterol levels and the incidence of ischemic changes in electrocardiograms. In cases of HDL cholesterol < 1.81 mmol/L (70 mg/dL), the incidence increased in proportion to the levels of HDL cholesterol. The frequency of the CETP gene mutation was higher in patients with coronary heart disease than in healthy control subjects. In subjects aged > 80 years, the prevalence of both marked HALP and the intron 14 splicing defect was significantly lower than in the younger generation. The current study indicated for the first time that a marked HALP caused by CETP gene mutation may not represent a longevity syndrome, suggesting the importance of reevaluation of the clinical significance and pathophysiology of a marked HALP.

Cholesteryl ester transfer protein activity enhances plasma cholesteryl ester formation. Studies in CETP transgenic mice and human genetic CETP deficiency.

The plasma cholesteryl ester transfer protein (CETP) promotes the removal of HDL cholesteryl esters and is thought to stimulate reverse cholesterol transport (RCT). However, mechanisms by which CETP may stimulate RCT are poorly understood. Thus, we examined the relationship between plasma CETP expression and plasma cholesteryl ester formation in CETP transgenic (Tg) mice, hamsters, and human subjects with genetic CETP deficiency. Incubation of CETP Tg mouse plasma showed a 20% to 40% increase in plasma cholesterol esterification rate (CER, P < .05) compared with control mice. Injection of a neutralizing CETP monoclonal antibody (MAb) (TP2) into natural flanking region CETP Tg mice resulted in an increase in plasma free cholesterol (FC) concentration, FC/CE ratio, FC/phosphatidylcholine ratio, and hepatic CETP mRNA. In hamsters, CETP inhibition also resulted in an increase in plasma FC/phosphatidylcholine ratio and increased CETP mRNA in adipose tissue. In humans with two common CETP gene mutations (an intron 14 splicing defect and a D442G missense mutation), mean plasma CERs were 39 and 60, respectively, compared with 89 nmol x mL-1 x h-1 in normal subjects. By contrast, lecithin:cholesterol acyltransferase (LCAT) mass was normal in CETP-deficient subjects. MAb neutralization of CETP activity in incubated human plasma did not alter the LCAT reaction, even after supplementation with discoidal HDL and VLDL. Thus, genetic alterations in CETP levels lead to secondary changes in the plasma LCAT reaction, possibly because of remodeling of HDL by CETP acting in concert with other factors in vivo. In human genetic CETP deficiency, a moderate impairment in the plasma LCAT reaction may contribute to a defect in RCT, providing a potential mechanism to explain the recently observed excess of coronary heart disease in these subjects.

Molecular genetics of plasma cholesteryl ester transfer protein

Plasma cholesteryl ester transfer protein facilitates the transfer of cholesteryl ester from HDL to apolipoprotein B-containing lipoproteins, and is a key protein in the reverse cholesterol transport system. The importance of plasma cholesteryl ester transfer protein in lipoprotein metabolism was highlighted by the discovery of deficient individuals with a marked hyper-HDL-cholesterolemia. Cholesteryl ester transfer protein deficiency causes various abnormalities in the concentration, composition, and functions of both HDL and LDL. Its significance in atherosclerosis is still controversial. However, in-vitro evidence shows large cholesteryl ester-rich HDL particles in cholesteryl ester transfer protein deficiency are defective in cholesterol efflux. Recent epidemiological studies have demonstrated an increased incidence of coronary atherosclerosis in deficient patients. The current review will also focus on the molecular genetics of the protein.

Exon 10 skipping caused by intron 10 splice donor site mutation in cholesteryl ester transfer protein gene results in abnormal downstream splice site selection

Cholesteryl ester transfer protein (CETP) deficiency is the most common cause of hyperalphalipoproteinemia in Japan. However, the genetic basis of this disorder has not been fully characterized. We have studied a 49-year-old Japanese male presenting with total cholesterol, HDL-cholesterol, and apolipoprotein A-I levels of 300, 236, and 233 mg/dl, respectively, and total absence of CETP activity and mass in plasma. Sequence analysis of the patient's CETP gene revealed that the splice donor consensus GT was substituted by GG in intron 10 (intron 10 splice defect) and by AT in intron 14 (intron 14 splice defect). Restriction digestion of PCR-amplified DNA using NdeI and MaeIII established that the patient was a compound heterozygote for both gene defects. Sequencing of cDNA amplified by RT-PCR from the patient's monocyte-derived macrophage RNA demonstrated abnormal splicing with deletion of exon 10 as well as alternative splicing at a native AG site located 31 nucleotides 5' of the normal splice acceptor in intron 13. Thus, the intron 10 splice defect results in exon 10 skipping and the insertion of a 31 bp fragment between exon 13 and exon 14, which contains an in frame stop codon. The presence of abnormally spliced mRNA was further confirmed by amplification of patient cDNA using CETP specific primers. Abnormal splicing of exon 14 as a result of the intron 14 splice defect was not detected, indicating potential unstable CETP mRNA derived from that mutation. These findings demonstrate that a novel splice site mutation in intron 10 of the CETP gene results in the skipping of exon 10, as well as disruption of downstream splicing at intron 13 identifying a novel mechanism leading to CETP deficiency.

A novel nonsense mutation (G181X) in the human cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects

Cholesteryl ester transfer protein (CETP) plays an important role in regulating the concentration and composition of high density lipoprotein (HDL) and low density lipoprotein (LDL). Although several genetic abnormalities causing CETP deficiency have been identified in the Japanese subjects with a marked hyperalphalipoproteinemia (HALP), there are many CETP-deficient subjects for whom the genetic abnormalities have not been clarified. In the present study, we analyzed the molecular basis of an HALP subject without CETP activity and mass, and found a novel mutation in the CETP gene. This novel mutation (G181X) was a G-to-T substitution at codon 181 of exon 6 which replaced a codon for glycine (GGA) with a premature stop codon (TGA). The G181X mutation created a new cutting site by restriction enzyme MaeIII. To estimate the frequency of G181X, we investigated unrelated 294 HALP (HDL-cholesterol > or = 2.59 mmol/L = 100 mg/dl) subjects by restriction fragment length polymorphism (RFLP) analysis with Mae III. One (0.34%) HALP subject was homozygous and four (1.36%) were heterozygous for this mutation. The allelic frequency of a G-to-T substitution at codon 181 of exon 6 was 0.0102 in HALP subjects. From the lipid analysis of the proband and the homozygote, it was clarified that the G181X mutation had dominant effects on HDL and LDL metabolism, similar to a G-to-A substitution at the 5' splice donor site of the intron 14 (1451 + 1G-->A). In conclusion, the G181X mutation is one of causes of HALP in the Japanese HALP subjects, having dominant effects on lipid metabolism.

Increased coronary heart disease in Japanese-American men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels.

Plasma high density lipoprotein (HDL) levels are strongly genetically determined and show a general inverse relationship with coronary heart disease (CHD). The cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters from HDL to other lipoproteins and is a key participant in the reverse transport of cholesterol from the periphery to the liver. A high prevalence of two different CETP gene mutations (D442G, 5.1%; intron 14G:A, 0.5%), was found in 3,469 men of Japanese ancestry in the Honolulu Heart Program and mutations were associated with decreased CETP (-35%) and increased HDL chol levels (+10% for D442G). However, the overall prevalence of definite CHD was 21% in men with mutations and 16% in men without mutations. The relative risk (RR) of CHD was 1.43 in men with mutations (P < .05); after adjustment for CHD risk factors, the RR was 1.55 (P = .02); after additional adjustment for HDL levels, the RR was 1.68 (P = .008). Similar RR values were obtained for the D442G mutation alone. Increased CHD in men with mutations was primarily observed for HDL chol 41-60 mg/dl; for HDL chol > 60 mg/dl men with and without mutations had low CHD prevalence. Thus, genetic CETP deficiency appears to be an independent risk factor for CHD, primarily due to increased CHD prevalence in men with the D442G mutation and HDL cholesterol between 41 and 60 mg/dl. The findings suggest that both HDL concentration and the dynamics of cholesterol transport through HDL (i.e., reverse cholesterol transport) determine the anti-atherogenicity of the HDL fraction.

Molecular disorders of cholesteryl ester transfer protein.

Plasma cholesteryl ester transfer protein (CETP) facilitates the transfer of cholesteryl ester (CE) from HDL to apolipoprotein B-containing lipoproteins and therefore is a key protein in the reverse cholesterol transport system. The importance of plasma CETP in lipoprotein metabolism has been highlighted by the discovery of CETP-deficient subjects with a marked hyper-HDL-cholesterolemia. The deficiency of CETP causes various abnormalities in the concentration, composition, and functions of high density and low density lipoproteins. The current review will focus on some of the recent knowledge on CETP with special reference to the biochemical and molecular biological aspects of CETP. Furthermore, detailed information will be presented regarding the lipoprotein abnormalities and molecular basis of CETP deficiency.

CETP(Cholesteryl Ester Transfer Protein) Deficiency Caused by Genetic Mutation in the CETP Gene in Normal Korean Population

CETP(Cholesteryl Ester Transfer Protein) Deficiency Caused by Genetic Mutation in the CETP Gene in Normal Korean Population

Cholesteryl ester transfer protein deficiency: Identification in the Chinese

Cholesteryl ester transfer protein (CETP) regulates cholesterol content in high-density lipoproteins (HDL) through the exchange of cholesteryl ester in HDL and triglyceride in triglyceride-rich lipoproteins. Three types of CETP deficiency have so far been reported, all in Japanese individuals. Among them, two types of mutation in the CETP gene are relatively common: (1) a guanine to adenine mutation in the intron 14 splicing donor site (114A), and (2) an Asp 442 to Gly (D442G) mutation. The 114A mutation has an estimated allele frequency of 0.81% in the Japanese. It elevates the plasma HDL-cholesterol level strikingly: typically, >120 mg/dl for homozygotes and 80-110 mg/dl for heterozygotes. In contrast, the D442G mutation has a higher estimated allele frequency (4.62%), but has a mild effect on the HDL-cholesterol level: typically, >80 mg/dl for homozygotes and 50-100 mg/dl for heterozygotes. We screened Chinese individuals for the two CETP gene mutations. 7 refs., 1 tab.

Evaluation of G-to-A substitution in the apolipoprotein A-I gene promoter as a determinant of high-density lipoprotein cholesterol level in subjects with and without cholesteryl ester transfer protein deficiency

The effect of a polymorphism, guanine (G) to adenine (A) substitution in the promoter of apolipoprotein A-I gene at a position 78 bp upstream of the transcription initiation site, on the serum high-density lipoprotein (HDL)-cholesterol level was studied in 168 Japanese subjects with HDL-cholesterol levels ranging from 26 to 171 mg/dl. Considering the significant effect of cholesteryl ester transfer protein (CETP) on the HDL-cholesterol level and the common occurrence of its deficiency, we performed statistical analyses separately for two groups: one without CETP deficiency (n = 126) and the other with CETP deficiency (n = 42). In the group without CETP deficiency, in which the numbers of G/G, G/A, and A/A genotypes were 92 (73.0%), 28 (22.2%), and 6 (4.8%), respectively, the frequency of the A allele in the subjects with HDL-cholesterol levels of > or = 70 mg/dl did not differ from subjects with HDL-cholesterol levels of < or = 69 mg/dl, irrespective of gender: 0.154 and 0.145 in males, and 0.182 and 0.174 in females, respectively, for the > or = 70 mg/dl and < or = 69 mg/dl groups. Additionally, the HDL-cholesterol levels for the subjects with the G/G genotype did not differ from those for the subjects with the A allele: 64 +/- 22, 58 +/- 14, 77 +/- 14 and 62 +/- 16 mg/dl, respectively, for the G/G, G/A, A/A, and G/A + A/A in males, and 72 +/- 18, 74 +/- 24, 63 +/- 4, and 73 +/- 23 mg/dl in females. For the group with CETP deficiency, in which the numbers of G/G and G/A + A/A genotypes were 25 (59.5%) and 17 (40.5%), the HDL-cholesterol levels also did not differ: 98 +/- 24 mg/dl and 99 +/- 30 mg/dl, respectively, for the G/G and G/A + A/A genotypes. Thus, there is no evidence that the polymorphism has any effect on serum HDL-cholesterol levels regardless of CETP status. We conclude that the G-to-A substitution in the promoter of apolipoprotein A-I gene does not significantly alter serum HDL-cholesterol level.

Atherosclerotic Disease in Marked Hyperalphalipoproteinemia

Hyperalphalipoproteinemia (HALP) has been regarded as a beneficial state accompanied by a longevity syndrome. However, we reported the cases of markedly hyperalphalipoproteinemic subjects with juvenile corneal opacification. The patients had reduced postheparin hepatic triglyceride lipase (HTGL) activities, and one of them has recently been identified to be homozygous for a missense mutation in exon 15 (D442: G) in the cholesteryl ester transfer protein (CETP) gene. In the current study, to elucidate the clinical significance of and atherogenicity in marked HALP, we determined the incidence of atherosclerotic cardiovascular disease (ACD) in patients with marked HALP and characterized the lipoprotein abnormalities in those who had ACD, focusing especially on CETP and HTGL. The subjects were 201 patients (111 males and 90 females) with marked HALP ( > or = 2.58 mmol/L [100 mg/dL]), 67% of whom were demonstrated to have the CETP gene mutations in the intron 14 splice donor site or in exon 15. Their mean age was 54 +/- 15 years. Plasma levels of total cholesterol, HDL cholesterol, and triglyceride in all subjects were 6.28 +/- 1.78, 3.15 +/- 0.90, and 1.08 +/- 0.53 mmol/L, respectively. Ten of the male patients (9.0%) and two of the female patients (2.2%) had apparent ACD such as myocardial infarction, angina pectoris, and peripheral vascular diseases. Ten patients with HALP who had ACD were identified to be heterozygotes for CETP deficiency. To further clarify the characteristics of marked HALP in patients with ACD, we compared the plasma lipids, lipoproteins, CETP, and HTGL activities between heterozygotes for CETP deficiency who were with and without ACD.

Cholesteryl ester transfer proteins, reverse cholesterol transport, and atherosclerosis.

Plasma cholesteryl ester transfer protein plays a central role in lipoprotein metabolism by exchanging cholesteryl esters with triglycerides. Human genetic deficiency is associated with increased HDL-cholesterol levels, whereas cholesteryl ester transfer protein overexpression in transgenic mice results in decreased HDL-cholesterol. Thus, it has been proposed that cholesteryl ester transfer protein deficiency is an antiatherogenic state. However, recent observations in human cholesteryl ester transfer protein deficiency and cholesteryl ester transfer protein transgenic mice also suggest antiatherogenic effects of the expression of this protein, probably reflecting its role in reverse cholesterol transport.

Increased catabolic rate of low density lipoproteins in humans with cholesteryl ester transfer protein deficiency.

The cholesteryl ester transfer protein (CETP) transfers lipids among lipoprotein particles and plays a central role in lipoprotein metabolism. Humans with genetic deficiency of CETP have both elevated HDL cholesterol and apolipoprotein A-I concentrations as well as decreased LDL cholesterol and apolipoprotein B levels. The present study was undertaken to elucidate the metabolic basis for the decreased LDL cholesterol and apo B levels in CETP deficiency. We conducted a series of in vivo apo B kinetic studies in tow unrelated homozygotes with CETP deficiency and in control subjects. A primed constant infusion of stable isotopically labeled phenylalanine was administered to the two CETP deficient subjects and control subjects and apo B kinetic parameters in VLDL, intermediate density lipoproteins, and LDL were obtained by using a multicompartmental model. The fractional catabolic rates (FCR) of LDL apo B were significantly increased in the CETP-deficient subjects (0.56 and 0.75/d) compared with the controls (mean FCR of 0.39/d). Furthermore, the production rates of apo B in VLDL and intermediate density lipoprotein were decreased by 55% and 81%, respectively, in CETP deficiency compared with the controls. In conclusion, CETP-deficient subjects were demonstrated to have substantially increased catabolic rates of LDL apo B as the primary metabolic basis for the low plasma levels of LDL apo B. This result indicates that the LDL receptor pathway may be up-regulated in CETP deficiency.

Decreased affinity of low density lipoprotein (LDL) particles for LDL receptors in patients with cholesteryl ester transfer protein deficiency

We have reported that the disorder of lipoprotein metabolism in hyperalphalipoproteinaemic patients with a deficiency of cholesteryl ester transfer protein (CETP) is characterized by the polydisperse low density lipoprotein (LDL) particles and the accumulation of cholesteryl ester (CE) in high density lipoprotein (HDL) particles, forming cholesterol-induced HDL (HDLc)-like particles. In the present study we have investigated the interaction of these abnormal LDL with LDL receptors of normal human fibroblasts. Since the ultracentrifugally separated LDL fraction (1.019 < d < 1.063 g mL-1) from the CETP-deficient patients contained HDLc-like particles, these particles were removed by anti-apolipoprotein (apo) A-I immunoaffinity column chromatography. The lipoproteins eluted in the unbound fraction of this column did not contain apo A-I, so this fraction was considered to be authentic LDL. The authentic LDL of the patients were deficient in CE and rich in triglycerides and apo B. The authentic LDL itself showed polydispersity, ranging in size from 23 nm to 30 nm. The affinity of these abnormal LDL particles for LDL receptors was analysed by a competitive assay in which cold LDL from the patients or control compete with 125I-labelled LDL for fibroblast LDL receptors. The concentration of LDL particles at which 50% of 125I-labelled normal LDL was replaced was two to three times higher for the patients than for the normal control. Therefore, the affinity of patient LDL was thought to be reduced compared to that of control LDL. These results demonstrate that CETP may play an important role in making LDL particles homogeneous and rich in CE.(ABSTRACT TRUNCATED AT 250 WORDS)