Familial lecithin-cholesterol acyltransferase: Identification of heterozygotes with half-normal enzyme activity and mass

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Canadian kindred of Italian and Swedish descent with familial LCAT deficiency. Four subjects had LCAT mass of 5.21±0.87 g/ml (mean±SD) and LCAT activity of 98.8±12.0 nmol/h/ml, well within their respective normal ranges. Five family members, including the parents, the maternal grandmother, and two of four siblings of the LCAT deficient subjects, had enzyme mass (2.85±0.32 g/ml) and activity (50.8±6.3 nmol/h/ml) approximately one-half that of normal levels. These presumed heterozygotes had normal levels of apolipoproteins A-I, A-II, B and D. The two subjects with LCAT deficiency had no detectable LCAT mass (below 0.1 g/ml) or LCAT activity (below 0.76 nmol/h/ml), apolipoprotein A-I and D levels approximately 50% of normal, and apolipoproteins B and A-II levels only 30–35% of normal. LCAT deficiency in this family is determined by an autosomal recessive mode. Furthermore, LCAT levels and activity are determined by two autosomal codominant alleles, LCATⁿ, the normal LCAT gene, and LCAT^d, the LCAT deficiency gene.     

Apolipoprotein AIMilano. Partial lecithin:cholesterol acyltransferase deficiency due to low levels of a functional enzyme

The cholesterol esterification process was analyzed in 19 carriers of the apolipoprotein AIMilano (AIM) variant and in 19 age-sex matched controls by measuring lecithin:cholesterol acyltransferase (LCAT) mass, activity (i.e., cholesterol esterification with a standard proteoliposome substrate) and cholesterol esterification rate (i.e., cholesterol esterification in the presence of the endogenous substrate). The AIM subjects had lower LCAT mass (3.30 +/- 0.85 micrograms/ml), activity (71.1 +/- 36.4 nmol/ml per h) and cholesterol esterification rate (23.6 +/- 12.5 nmol/ml per h) compared to controls (5.22 +/- 0.74 micrograms/ml, 121.6 +/- 54.6 nmol/ml per h and 53.6 +/- 29.9 nmol/ml per h, respectively). The specific LCAT activity, i.e., LCAT activity per microgram of LCAT, was similar in the two groups, indicating that the LCAT protein in the AIM carriers is structurally and functionally normal. However, the specific cholesterol esterification rate was 23% lower in the AIM subjects (8.03 +/- 6.01 nmol/h per microgram) compared to controls (10.49 +/- 5.86 nmol/h per microgram; P less than 0.05). The capacity of HDL3, purified from both AIM and control plasma, to act as substrates for cholesterol esterification was similar, thus suggesting that other mechanism(s) may be in play. Carriers with a relative abundance of abnormal, small HDL3b particles had the most altered cholesterol esterification pattern. Upon evaluating all AIM subjects, a complex relationship between HDL structure, plasma lipid-lipoprotein levels and cholesterol esterification emerged, making the AIMilano condition a unique model for the study of the mechanisms regulating the cholesterol esterification-transfer process in man.     

Detection of heterozygotes for familial lecithin: cholesterol acyltransferase (LCAT) deficiency.

"Rocket" immunoelectrophoresis using specific anti-lecithin: cholesterol acyltransferase (LCAT) antiserum showed no immunoreactive protein in two patients with familial LCAT deficiency. Subnormal quantity of plasma LCAT was found in the maternal grandmother, the parents, and in two of four siblings of the patients (3.3-3.4 mg/l vs. 5.4 +/- 0.5 mg/l in 12 controls). The immunochemical quantitation of the enzyme correlated well (r = .93) with LCAT activity in an artificial substrate assay. These two methods allow detection of heterozygotes for LCAT deficiency.     

ApoA-I(MALLORCA) impairs LCAT activation and induces dominant familial hypoalphalipoproteinemia.

Apolipoprotein (apo)A-I is the major protein component of HDL and the cofactor for LCAT. We describe a large Spanish kindred, living in the Mediterranean Island of Mallorca, that presents a dominant form of hypoalphalipoproteinemia. The lipid profile of this family was studied because the proband, a 40-year-old male presenting signs of coronary atherosclerosis, showed severe HDL deficiency. However, none of the other family members had a known history of cardiovascular disease. Sequence analysis of the apoA-I gene in affected members identified a 33-base pair deletion, corresponding to residues 165-175 of the mature protein, eliminating the first 11 amino acids of the internal repeat 7. ApoA-I(MALLORCA) is associated with HDL-cholesterol deficiency (concentration ranging from 8-48% of the value in non-carriers), and a 2- to 3-fold decrease in plasma concentrations of apoA-I and apoA-II and endogenous LCAT activity, concomitant with a slight decrease in serum cholesterol efflux capability. Impairment of LCAT activity in HDL particles containing only mutated forms of apoA-I would not explain a pattern of dominant inheritance. HDL particles containing wild type apoA-I and at least one mutant apoA-I may also present impaired LCAT activity and/or other alterations leading to defective HDL maturation, a situation that would increase HDL lipid catabolism.We conclude that amino acids 165-175 of apoA-I are critical for normal HDL metabolism, at least in part because of their role in LCAT activation. However, apoA-I(MALLORCA) is not necessarily associated with clinical signs of atherosclerosis.     

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.     

Familial lecithin-cholesterol acyltransferase deficiency in a Japanese family: Evidence for functionally defective enzyme in homozygotes and obligate heterozygotes

Summary Lecithin-cholesterol acyltransferase (LCAT) mass and activity were measured in a Japanese family with familial LCAT deficiency. The two LCAT-deficient subjects had LCAT mass approximately 40–46% of normal (2.65 and 2.31 g/ml respectively, as compared with normal levels of 5.76±0.95 g/ml in 19 Japanese subjects) and enzyme activity less than 10% of normal (9.1 and 8.3 nmol/h/ml respectively, as compared with normal levels of 100 nmol/h/ml). All obligate heterozygotes examined, including the father of the two LCAT-deficient subjects, and all five children of the deficient subjects had LCAT mass approximately 72–80% of the normal LCAT mass (4.12, 4.38, 4.45, 4.48, 4.49, 4.61 g/ml, respectively) and LCAT activity approximately half normal (51.9, 52.4, 54.2, 56.6, and 57.2 nmol/h/ml). We conclude that the two LCAT-deficient subjects of this family have functionally defective enzyme. Furthermore, the data suggest that the plasma of the obligate heterozygotes contain both normal and functionally defective enzymes.     

Lecithin-cholesterol acyltransferase (LCAT) deficiency with a missense mutation in exon 6 of the LCAT gene

The plasma enzyme, human lecithin-cholesterol acyltransferase (LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.     

Recombinant human LCAT normalizes plasma lipoprotein profile in LCAT deficiency

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for cholesterol esterification in plasma. Mutations in the LCAT gene leads to two rare disorders, familial LCAT deficiency and fish-eye disease, both characterized by severe hypoalphalipoproteinemia associated with several lipoprotein abnormalities. No specific treatment is presently available for genetic LCAT deficiency. In the present study, recombinant human LCAT was expressed and tested for its ability to correct the lipoprotein profile in LCAT deficient plasma. The results show that rhLCAT efficiently reduces the amount of unesterified cholesterol (?30%) and promotes the production of plasma cholesteryl esters (+210%) in LCAT deficient plasma. rhLCAT induces a marked increase in HDL-C levels (+89%) and induces the maturation of small preβ-HDL into alpha-migrating particles. Moreover, the abnormal phospholipid-rich particles migrating in the LDL region were converted in normally sized LDL.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.     

A single G to A nucleotide transition in exon IV of the lecithin: cholesterol acyltransferase (LCAT) gene results in an Arg140 to His substitution and causes LCAT-deficiency

We have characterized the molecular defect causing lecithin:cholesterol acyltransferase (LCAT)-deficiency (LCAT-D) in the LCAT gene in three siblings of Austrian descent. The patients presented with typical symptoms including corneal opacity, hemolytic anemia, and kidney dysfunction. LCAT activities in the plasma of these three patients were undetectable. DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA of all six LCAT exons revealed a new point mutation in exon IV of the LCAT gene, i.e., a G to A substitution in codon 140 converting Arg to His. This mutation caused the loss of a cutting site for the restriction endonuclease HhaI within exon IV: Upon digestion of a 629-bp exon IV PCR product with HhaI, the patients were found to be homozygous for the mutation. Eight of 11 family members were identified as heterozygotes. Transfection studies of COS-7 cells with plasmids containing a wildtype or a mutant LCAT cDNA revealed that, in contrast to the cell medium containing wild-type enzyme, no enzyme activity was detectable upon expression of the mutant protein. This represents strong evidence for the causative nature of the observed mutation for LCAT deficiency in affected individuals and supports the conclusion that Arg¹⁴⁰ is crucial for the structure of an enzymatically active LCAT protein.     

遗传性LCAT缺陷症与动脉粥样硬化

卵磷脂：胆固醇酰基转移酶（lecithin：cholesterol acyltransferase,LCAT）参与胆固醇酯的合成并在高密度脂蛋白（high density lipoprotein,HDL）的代谢中起重要作用。遗传性LCAT缺陷症是一种以低HDL-胆固醇（HDL-C）为特点的罕见遗传疾病。近年来,LCAT在HDL-C代谢中以及在动脉粥样硬化发生和发展中的作用逐渐被本领域研究者所关注。本文就LCAT缺陷症的遗传学和生化学特点做一综述,重点阐述为何尽管HDL-C水平明显减低,LCAT突变携带者却并未发生早期动脉粥样硬化。     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

Effect of LCAT on HDL-mediated cholesterol efflux from loaded EA.hy 926 cells

• 1.1. Human endothelial cells (EA.hy 926 line) were loaded with cholesterol, using cationized LDL, and the effect of lecithin:cholesterol acyltransferase (LCAT) on cellular cholesterol efflux mediated by high density lipoproteins (HDL) was measured subsequently.
• 2.2. In plasma, lecithin:cholesterol acyltransferase (LCAT) converts unesterified HDL cholesterol into cholesteryl esters, thereby maintaining the low UC/PL ratio of HDL. It was tested if further decrease in UC/PL ratio of HDL by LCAT influences cellular cholesterol efflux in vitro.
• 3.3. Efflux was measured as the decrease of cellular cholesterol after 24 hr of incubation with various concentrations of HDL in the presence and absence of LCAT. LCAT from human plasma (about 3000-fold purified) was added to the cell culture, resulting in activity levels in the culture media of 60–70% of human serum.
• 4.4. Although LCAT had a profound effect on HDL structure (UC/TC and UC/PL ratio's decreased), the enzyme did not enhance efflux of cellular cholesterol, using a wide range of HDL concentrations (0.05–2.00 mg HDL protein/ml).
• 5.5. The data indicate that the extremely low unesterified cholesterol content of HDL, induced by LCAT, does not enhance efflux of cholesterol from loaded EA.hy 926 cells. It is concluded that the HDL composition (as isolated from plasma by ultracentrifugation) is optimal for uptake of cellular cholesterol.

     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Changes in serum lipids and lecithin: cholesterol acyltransferase enzyme during 1 week weight reduction of women on a low-calorie diet

In 32 women of normal body weight who volunteered to participate in the study, the effect of rapid weight reduction by a low-calorie liquid diet on serum lipids and lecithin:cholesterol acyltransferase (LCAT) enzyme activity was studied. Women were on this 400 kJ/day diet for 7 days and fasting blood samples were drawn before and immediately after the diet. Serum cholesterol decreased from 5.7 +/- 1.0 to 5.2 +/- 1.1 mmol/l and high density lipoprotein cholesterol from 1.77 +/- 0.43 to 1.50 +/- 0.35 mmol/l. The serum LCAT activity decreased significantly during the weight reduction period. When serum LCAT activity was correlated to lipid parameters, a positive correlation was found with total cholesterol and triglyceride concentrations before weight reduction and also between changes in LCAT activity and total cholesterol concentration. The data suggest that serum LCAT activity might have a prominent role in the regulation of serum lipid levels.     

Effect of mutations of N- and C-terminal charged residues on the activity of LCAT

On the basis of structural homology calculations, we previously showed that lecithin:cholesterol acyltransferase (LCAT), like lipases, belongs to the alpha/beta hydrolase fold family. As there is higher sequence conservation in the N-terminal region of LCAT, we investigated the contribution of the N- and C-terminal conserved basic residues to the catalytic activity of this enzyme. Most basic, and some acidic residues, conserved among LCAT proteins from different species, were mutated in the N-terminal (residues 1;-210) and C-terminal (residues 211;-416) regions of LCAT. Measurements of LCAT-specific activity on a monomeric substrate, on low density lipoprotein (LDL), and on reconstituted high density lipoprotein (rHDL) showed that mutations of N-terminal conserved basic residues affect LCAT activity more than those in the C-terminal region. This agrees with the highest conservation of the alpha/beta hydrolase fold and structural homology with pancreatic lipase observed for the N-terminal region, and with the location of most of the natural mutants reported for human LCAT. The structural homology between LCAT and pancreatic lipase further suggests that residues R80, R147, and D145 of LCAT might correspond to residues R37, K107, and D105 of pancreatic lipase, which form the salt bridges D105-K107 and D105-R37. Natural and engineered mutations at residues R80, D145, and R147 of LCAT are accompanied by a substantial decrease or loss of activity, suggesting that salt bridges between these residues might contribute to the structural stability of the enzyme.     

Effect of etiroxate on LCAT activity and on certain energy metabolism enzymes in the rat

Etiroxate (Skleronorm Grünenthal R) was administered 42 days to male Wistar rats and their serum and liver cholesterol and triglyceride levels, the rate of esterification of free cholesterol in their plasma by lecithin cholesterol acyltransferase (LCAT) (EC 2.3.1.43) and thriosephosphate dehydrogenase (TPDH) (EC 1.2.1.12), lactate dehydrogenase (LDH) (EC 1.1.1.27), hexokinase (HK) (EC 2.7.1.1), c-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), malate dehydrogenase (MDH) (EC 1.1.1.37) citrate synthase (CS) (EC 4.1.3.7) and hydroxyacylcoenzyme A dehydrogenase (HOADH) (EC 1.1.1.35) activity were determined in their liver. After 14 and 28 days, animals given etiroxate (600 micrograms/kg) had smaller weight increments than the controls and a significantly lower plasma free and esterified cholesterol level, but a significantly higher liver cholesterol concentration. Their final plasma and liver cholesterol concentrations did not differ significantly from the control values. Plasma triglyceride levels were significantly raised in treated animals at all the given intervals. LCAT activity was significantly higher throughout the whole time of treatment, with the maximum increase in the last phase. Glycolytic and oxidative enzyme activities were significantly raised, whereas GPDH activity was the same as in the controls. The results show that etiroxate accelerates cholesterol turnover in the endogenous pool by activating LCAT and stimulating energy metabolism.     

LCAT can rescue the abnormal phenotype produced by the natural ApoA-I mutations (Leu141Arg)Pisa and (Leu159Arg)FIN

To explain the etiology and find a mode of therapy of genetically determined low levels of high-density lipoprotein (HDL), we have generated recombinant adenoviruses expressing apolipoprotein A-I (apoA-I)(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN and studied their properties in vitro and in vivo. Both mutants were secreted efficiently from cells but had diminished capacity to activate lecithin/cholesterol acyltransferase (LCAT) in vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA-I-deficient (apoA-I-/-) mice resulted in greatly decreased total plasma cholesterol, apoA-I, and HDL cholesterol levels. The treatment also decreased the cholesteryl ester to total cholesterol ratio (CE/TC), caused accumulation of prebeta1-HDL and small size alpha4-HDL particles, and generated only few spherical HDL particles, as compared to mice expressing wild-type (WT) apoA-I. Simultaneous treatment of the mice with adenoviruses expressing either of the two mutants and human LCAT normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal prebeta- and alpha-HDL subpopulations, and generated spherical HDL. The study establishes that apoA-I(Leu141Arg)Pisa and apoA-I(Leu159Arg)FIN inhibit an early step in the biogenesis of HDL due to inefficient esterification of the cholesterol of the prebeta1-HDL particles by the endogenous LCAT. Both defects can be corrected by treatment with LCAT.     

Novel P143L polymorphism of the LCAT gene is associated with dyslipidemia in Chinese patients who have coronary atherosclerotic heart disease

Coronary atherosclerotic heart disease (CAD) is a multifactorial disorder resulting from numerous gene-gene and gene-environment interactions. Lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport and the metabolism of high-density lipoprotein (HDL), is thought to be a candidate gene related to dyslipidemia and CAD. Variations in the LCAT gene were investigated in 190 CAD patients and 209 age- and gender-matched controls by denaturing high-performance liquid chromatography, and confirmed by sequencing and RFLP assay. In CAD patients, a novel single-nucleotide polymorphism (P143L) in exon 4 of the LCAT gene was discovered in nine males and two females (frequency of 5.79%), which was found in none of 209 controls. The genotype and allele distribution of P143L is significantly (P<0.04 ) higher in the low HDL-C subgroup than in the normal HDL-C subgroup in both male patients and all CAD patients. P143L was also found to be significantly (P<0.01) associated with the low HDL-C phenotype in both male patients and all CAD patients, with odds-ratios of 7.003 (95% CI 2.243-21.859) and 5.754 (95% CI 1.893-13.785), respectively. Thus, the P143L polymorphism may play a role in causing decreased HDL-C levels, leading to increased risk of dyslipidemia and CAD in Chinese.