High plasma HDL concentrations associated with enhanced atherosclerosis in transgenic mice overexpressing lecithin-cholesteryl acyltransferase.

A subset of patients with high plasma HDL concentrations have enhanced rather than reduced atherosclerosis. We have developed a new transgenic mouse model overexpressing human lecithin-cholesteryl acyltransferase (LCAT) that has elevated HDL and increased diet-induced atherosclerosis. LCAT transgenic mouse HDLs are abnormal in both composition and function. Liver uptake of [3H]cholesteryl ether incorporated in transgenic mouse HDL was reduced by 41% compared with control HDL, indicating ineffective transport of HDL-cholesterol to the liver and impaired reverse cholesterol transport. Analysis of this LCAT-transgenic mouse model provides in vivo evidence for dysfunctional HDL as a potential mechanism leading to increased atherosclerosis in the presence of high plasma HDL levels.

Donor splice site mutation in the apolipoprotein (Apo) C-II gene (Apo C-IIHamburg) of a patient with Apo C-II deficiency.

The DNA, RNA, and protein of apo C-II have been analyzed in a patient with apo C-II deficiency (apo C-IIHamburg). Markedly reduced levels of plasma and intrahepatic C-II apolipoprotein were demonstrated by immunoblotting and immunohistochemical analysis. Northern, slot blot, and in situ hybridization studies revealed low levels of a normal-sized apo C-II mRNA. No major rearrangement of the apo C-II gene was detected by Southern blotting. Sequence analysis of apo C-II genomic clones revealed a G-to-C substitution within the donor splice site of intron II. This base substitution resulted in the formation of a new Dde I and loss of a Hph I restriction enzyme cleavage site. Amplification of the mutant sequence by the polymerase chain reaction and digestion with Dde I and Hph I restriction enzymes established that the patient was homozygous for the G-to-C mutation. This is the initial report of the DNA sequence of an abnormal apo C-II gene from a patient with deficiency of apo C-II. We propose that this donor splice site mutation is the primary genetic defect that leads to defective splicing and ultimately to an apo C-II deficiency in this kindred.

A nonsense mutation in the apolipoprotein C-IIPadova gene in a patient with apolipoprotein C-II deficiency.

The apo C-II gene from a patient with apo C-II deficiency has been sequenced after amplification by the polymerase chain reaction. A substitution of an adenosine for a guanosine at position 3002 in exon 3 of the patient's gene was identified by sequence analysis. This mutation leads to the introduction of a premature termination codon (TAA) at a position corresponding to amino acid 37 of mature apo C-II and to the formation of a new Rsa I restriction enzyme site not present in the normal apo C-II gene. Amplification of DNA from family members by the polymerase chain reaction and digestion with Rsa I established that the patient is a true homozygote for the mutation. Analysis of the patient's plasma by two-dimensional gel electrophoresis and immunoblotting detected an apo C-II that exhibited abnormal electrophoretic mobility. We propose that the C to A substitution in the apo C-IIPadova gene is the primary genetic defect that leads to premature termination and the synthesis of a truncated 36 amino acid apo C-II that is unable to activate lipoprotein lipase.

Fish eye syndrome: a molecular defect in the lecithin-cholesterol acyltransferase (LCAT) gene associated with normal alpha-LCAT-specific activity. Implications for classification and prognosis.

We have identified the molecular defect in two siblings presenting with classical clinical and biochemical features of Fish Eye disease (FED), including corneal opacities, HDL cholesterol < 10 mg/dl, normal plasma cholesteryl esters, and elevated triglycerides. In contrast to previously reported patients with FED who are unable to esterify HDL-associated cholesterol, our patients' plasma lecithin-cholesterol acetyltransferase (alpha-LCAT)-specific activities assayed using an HDL-like proteoliposome substrate were 12.7-25.7 nmol/micrograms (19.5 +/- 1.8 in controls). In addition, significant residual cholesterol esterification was present in VLDL/LDL-depleted plasma, confirming the presence of HDL-associated alpha-LCAT activity. DNA sequence analysis of the proband's LCAT gene identified deletion of the triplet coding for leu300, which resulted in the loss of a restriction site for MlnI. Digestion of PCR-amplified DNA using MlnI established that both siblings are homozygous for this defect. Expression of LCAT300-del. in human embryonic kidney-293 cells revealed normal mRNA and intracellular LCAT concentrations. However, reduced amounts of LCAT300-del., which had a normal specific alpha-LCAT activity, were present in the media. In summary, we report the first case of FED associated with a mutant enzyme that has a normal alpha-LCAT-specific activity. The functional significance of this LCAT gene defect has been established in an in vitro expression system, which demonstrates that very small amounts of this functional LCAT mutant enzyme accumulate in the media. Characterization of LCAT300-del. established that selective alpha-LCAT deficiency is not a prerequisite for the development of FED. On the basis of our combined results, we propose that the residual amounts of total plasma LCAT activity and not its distribution on lipoproteins primarily determines the heterogeneity in phenotypic expression observed in familial LCAT deficiency syndromes.

Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.

Apolipoprotein E (apoE)-deficient mice develop marked hyperlipidemia as well as atherosclerosis and thus are an excellent animal model for evaluating the potential for gene therapy in human genetic dyslipoproteinemias. Recombinant adenovirus containing either human apoE (rAdv.apoE) or the reporter gene luciferase (rAdv.luc) were generated and infused intravenously in apoE-deficient mice with preinfusion plasma total cholesterol of 644 +/- 149 mg/dl an cholesterol rich VLDL/IDL. After a single infusion of rAdv.apoE, plasma concentrations of human apoE ranging from 1.5 to 650 mg/dl were achieved. Adenovirus-mediated apoE replacement resulted in normalization of the lipid and lipoprotein profile with markedly decreased total cholesterol (103 +/- 18mg/dl), VLDL, IDL, and LDL, as well as increased HDL. Measurement of aortic atherosclerosis 1 mo after adenoviral infusion demonstrated a marked reduction in the mean lesion area of mice infused with rAdv.apoE (58 +/- 8 x 10(3) microns2) when compared with control mice infused with rAdv.luc (161 +/- 10 x 10(3) microns2; P < 0.0001). Thus, apoE expression for 4 wk was sufficient to markedly reduce atherosclerosis, demonstrating the feasibility of gene therapy for correction of genetic hyperlipidemias resulting in atherosclerosis. The combined use of adenovirus vectors and the apoE-deficient mouse represents a new in vivo approach that will permit rapid screening of candidate genes for the prevention of atherosclerosis.

Hyperalphalipoproteinemia in human lecithin cholesterol acyltransferase transgenic rabbits. In vivo apolipoprotein A-I catabolism is delayed in a gene dose-dependent manner.

Lecithin cholesterol acyltransferase (LCAT) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P<0.01) and hLCAT activity (r=-0.816, P<0.01). These data indicate that fractional catabolic rate is the predominant mechanism by which hLCAT overexpression differentially modulates HDL concentrations in this animal model. We hypothesize that LCAT-induced changes in HDL composition and size ultimately reduce apoA-I catabolism by altering apoA-I conformation and/or HDL particle regeneration.

Hepatic lipase gene therapy in hepatic lipase-deficient mice. Adenovirus-mediated replacement of a lipolytic enzyme to the vascular endothelium.

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.

ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice.

The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.

The isolation and characterization of a colony stimulating factor from human lung.

Serum-free conditioned medium from human lung obtained at autopsy provides a rich source of colony stimulating factor which stimulates granulocytic and macrophagic colony growth in both mouse and human bone marrow. The appearance of the factor is enhanced by endotoxin and inhibited by either puromycin or actinomycin D. Human lung colony stimulating factor is stable at the pH range of 6.5-10 and temperature of 56 degrees C for 30 min. It is resistant to trypsin and neuraminidase but is sensitive to subtilisin, chymotrypsin and periodate. It shows heterogeneity on Sephadex gel filtration with two activity peaks having molecular weight of 200 000 and 40 000, respectively. Upon gel electrophoresis, human lung colony stimulating factor migrates in the alpha-globulin post-albumin region. Using the combination procedures of hydroxyapatite chromatography and preparative polyacrylamide gel electrophoresis a 600-fold purification was achieved with a final specific activity of 6-10(5) units per mg protein. The purified colony stimulating factor is very labile; however, the activity can be stabilized by the addition of gelatin or bovine serum albumin at the concentration of 0.1% and 0.2 mg/ml, respectively.

Homozygous hypobetalipoproteinemia: transcriptional regulation and 5'-flanking sequence analysis in an apolipoprotein B deficiency state.

Apolipoprotein (apo) B is the principal apolipoprotein of chylomicrons, very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Patients with homozygous hypobetalipoproteinemia (HBL), characterized by apoB deficiency, have markedly decreased levels of hepatocyte mRNA as well as intracellular B apolipoprotein, and a virtual absence of plasma apoB. We have cloned, sequenced and analyzed the 5' regulatory region of the human apoB gene from -899 to +121 bp in normal and hypobetalipoproteinemic subjects. TATA and CAAT boxes were located at -30 and -61, respectively, and two GC-like boxes were identified at positions +56 and +108. The analysis of the HBL sequence revealed two substitutions at positions -838 and -517, when compared to the normal sequence. These substitutions were not present in any known apoB regulatory elements. The transcriptional activities of the homozygous hypobetalipoproteinemic and normal regulatory regions were compared by chloramphenicol acetyltransferase (CAT) assays in Hep G2 cells, and were found to be the same. Therefore, we conclude that the 5' regulatory region of the HBL apoB gene in this kindred is normal, and the two base substitutions do not affect promoter activity of the apoB gene. These studies suggest that a coding region abnormality in the apoB gene may lead to HBL.

Absence of triglyceride accumulation in lipoprotein lipase-deficient human monocyte-macrophages incubated with human very low density lipoprotein.

Lipoprotein lipase, a lipolytic enzyme essential for normal hydrolysis of triglycerides in very low density lipoprotein (VLDL) and chylomicrons, is found in several cell types, including macrophages. The role of lipoprotein lipase in mediating the uptake of normal VLDL triglycerides into human cultured monocyte-derived macrophages was studied using macrophage cells from a functionally lipoprotein lipase-deficient patient and macrophages of cells from a normal subject. After incubation with VLDL, massive accumulation of phase refractile (lipid) inclusions were noted by phase contrast microscopy within the normal, but not within the lipoprotein lipase-deficient, macrophages. Chemical determinations of intracellular lipid confirmed massive triglyceride accumulation within normal macrophages, but not in lipoprotein lipase-deficient macrophages. VLDL-derived cholesterol did not accumulate in either cell. These results confirm an additional role of lipoprotein lipase, that of mediating triglyceride accumulation into macrophages from normal human VLDL. Human monocyte-macrophages genetically deficient in a functional lipoprotein lipase will be useful to determine the role of lipoprotein lipase in macrophage accumulation of lipid from other forms of triglyceride-carrying lipoproteins, including hypertriglyceridemic VLDL, beta-VLDL, and chylomicrons.

The human preproapolipoprotein C-II gene. Complete nucleic acid sequence and genomic organization.

The complete nucleic acid sequence of human preproapolipoprotein (apo) C-II has been determined from 2 apoC-II clones isolated from 2 different human genomic DNA libraries. The cloned fragments were approx. 14 and 18 kb long, and sequence analysis established that the apoC-II gene consists of 3338 nucleotides containing 3 intervening sequences of 2391, 167, and 298 bases. The first intron is located within the 5'-untranslated region of apoC-II and contains 4 Alu type sequences. The second intron interrupts the codon specifying amino acid - 11 of the apoC-II signal peptide. The last intron, which contains a 38 bp sequence which is repeated 6 times, interrupts the codon specifying for amino acid +44 of the mature apolipoprotein.

The familial chylomicronemia syndrome.

The chylomicronemia syndrome is a disorder characterized by severe hypertriglyceridemia and fasting chylomicronemia. Genetic causes of the syndrome are rare and include deficiency of lipoprotein lipase (LPL), apolipoprotein C-II, and familial inhibitor of LPL. Patients with familial forms of hypertriglyceridemia in combination with secondary acquired disorders account for most individuals presenting with chylomicronemia. The clinical manifestations--lipid and other biochemical abnormalities--as well as treatment options for chylomicronemic patients are discussed.

Effectiveness of mevinolin on plasma lipoprotein concentrations in type II hyperlipoproteinemia.

Patients with low-density lipoprotein (LDL) concentrations in the top 10th percentile of the population (type II hyperlipoproteinemia [HLP]) are at increased risk for premature cardiovascular disease; however, the incidence of myocardial infarction and death can be decreased by LDL cholesterol reduction. Mevinolin, an inhibitor of endogenous cholesterol synthesis, has been shown to reduce LDL cholesterol concentrations in a subset of type II patients with heterozygous familial hypercholesterolemia (FH). Using a double-blind, randomized, crossover, placebo-controlled trial, the safety and efficacy of mevinolin were compared in 24 patients with type II HLP with heterozygous FH (n = 6) or without FH type II HLP (n = 18). Compared with placebo treatment, both apolipoprotein B and LDL cholesterol levels were reduced (p less than 0.01) in both FH and non-FH patients by 28 to 34% with mevinolin treatment. In addition, high-density lipoprotein cholesterol levels were significantly increased (p less than 0.001) in both patients with FH (16%) and those with non-FH type II HLP (14%). Patients had no serious or clinically significant adverse effects. Thus, mevinolin is a useful drug for treatment of most patients with elevated plasma LDL cholesterol concentrations.

Adenoviral delivery of low-density lipoprotein receptors to hyperlipidemic rabbits: receptor expression modulates high-density lipoproteins.

Plasma concentrations of low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) are inversely related in several dyslipoproteinemias. To elucidate the interactions between these lipoproteins, we used a recombinant adenovirus (hLDLR-rAdV) to express human LDL receptors (hLDLRs) in LDL receptor-deficient rabbits. hLDLR-rAdV administration resulted in hepatocyte expression and a reduction of total, intermediate-density lipoprotein (IDL), and LDL cholesterol. In addition, we found that hLDLR-rAdV treatment induced (1) increased very-low-density lipoprotein (VLDL) cholesterol, (2) increased VLDL, IDL and LDL triglycerides, (3) decreased alpha- and pre-beta-migrating apolipoprotein E (apo E) and decreased pre-beta-migrating apo A-I at 2 to 4 days posttreatment, and (4) increased total plasma apo A-I and pre-beta-migrating apo A-I beginning 8 to 10 days posttreatment. Virtually all plasma apo A-I was present on alpha- and pre-beta-HDL. Pre-beta-HDL particles with size and electrophoretic properties consistent with nascent HDL demonstrated the greatest relative apo A-I enrichment following hLDLR-rAdV treatment. In summary, enhanced expression of hepatocyte LDLRs by hLDLR-rAdV treatment markedly altered apo A-I-containing lipoproteins and IDL and LDL. The use of recombinant viruses to express physiologically relevant genes in intact animals, analogous to transfection of cells in culture, provides a new strategy for the evaluation of effects of specific gene products on metabolic systems in vivo.

A normal rate of cellular cholesterol removal can be mediated by plasma from a patient with familial lecithin-cholesterol acyltransferase (LCAT) deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme involved in the esterification of cholesterol in circulating plasma lipoproteins. In the present study, we describe the molecular defects in the LCAT gene and in lipoprotein metabolism of a 34-year-old patient presenting with features of classic familial LCAT deficiency. DNA sequencing revealed two separate point mutations in exon 3 of the patient's LCAT gene: a C to A substitution converting Tyr(83) to a Stop and a C to T transition converting an Arg(99) to a Cys. Digestion of patient PCR-amplified DNA with the restriction enzymes AccI and AciI established that the patient was a compound heterozygote for both mutations. In vitro expression of LCAT (Arg(99)-->Cys) in human embryonic kidney-293 cells demonstrated reduced expression, as well as reduced secretion and/or increased intracellular degradation of the mutant enzyme with significantly decreased alpha-LCAT specific activity, thus, establishing the functional significance of the LCAT (Arg(99)-->Cys) mutation. The plasma cholesterol esterification rate (CER, 2+/-0.3 nmol/ml/h), alpha-LCAT activity (2.9+/-0.1 nmol/ml/h) and LCAT concentration (0.3+/-0.1 microg/ml) were 2.9%, 2.3% and 6.1% that of normal subjects, respectively. Analysis of the patient's plasma lipid profile revealed reduced plasma concentrations of total cholesterol (111+/-0.5 mg/dl), HDL cholesterol (1.6+/-0.2 mg/dl), apolipoprotein (apo) A-I (52+/-4 mg/dl) and apo A-II (11+/-0.5 mg/dl). Nevertheless, for the first time, we demonstrate that the LCAT-deficient plasma is as efficient as control plasma in cholesterol efflux experiments performed with [(3)H]-cholesterol loaded fibroblasts. This result could explain the absence of premature atherosclerosis in this LCAT-deficient patient.

The molecular biology of human apoA-I, apoA-II, apoC-II and apoB.

The application of molecular biology techniques has enabled us to determine the gene sequence, organization, transcription and processing of apolipoprotein genes. Consequently, new insights have been gained in the biosynthesis and processing of these proteins. In addition to apoA-I, apoA-II and apoC-III reported here, other apolipoprotein genes such as apoC-II and apoE genes were found to share common intron-exon organizations. The results suggest that these genes most probably arise from a common ancestral gene. Utilizing cDNA as hybridization probes, we have localized apoA-I, apoA-II, apoC-II, apoC-III, apoE and apoB to specific locations of individual chromosomes (for review, see ref. 6). There is no clear relationship between currently known physiological function and the organization of the apolipoproteins in the chromosomes with the exception of the LDL receptor and its ligand, apoE which are localized to chromosome 19. However, apoB-100, the major ligand for the LDL receptor is on chromosome 2 and not in synteny with the apoE and the LDL receptor genes. The cloning of the major human apolipoprotein genes have also allowed us to initiate studies on the molecular defects leading to various dyslipoproteinemias including Tangier disease and abetalipoproteinemia. Undoubtedly, information derived from these studies will provide the basis for future in vitro and in vivo studies on patients with dyslipoproteinemia and premature atherosclerosis.

Role of hepatic and lipoprotein lipase in lipoprotein metabolism and atherosclerosis: studies in transgenic and knockout animal models and somatic gene transfer.

Hepatic lipase (HL) and lipoprotein lipase (LPL) are the two major lipolytic enzymes responsible for the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Both lipases are attached to the vascular endothelium via cell surface proteoglycans. HL is primarily involved in the metabolism of chylomicron remnants, intermediate density lipoproteins and high-density lipoproteins whereas LPL catalyzes the hydrolysis of triglycerides from chylomicrons and very low-density lipoproteins. In addition to their traditional function as lipolytic enzymes, HL and LPL appear to serve as ligands that mediate the interaction of lipoproteins to cell surface receptors and/or proteoglycans. Over the past several years significant advances have been made in our understanding of new, alternative mechanisms by which HL and LPL modulate lipoprotein metabolism and the development of atherosclerosis in vivo. This review will summarize some of the new insights generated from the study of transgenic and knockout HL and LPL animal models as well as somatic gene transfer of these two lipases.

Hypertriglyceridaemia due to genetic defects in lipoprotein lipase and apolipoprotein C-II.

Hypertriglyceridaemia, as defined by fasting triglyceride levels of greater than 2.8 mmol l-1, is a prevalent dyslipoproteinaemia in our population. The underlying pathophysiological mechanisms that result in elevations of plasma triglycerides are heterogeneous and, in most cases, incompletely understood. However, in a subset of patients presenting with this lipid disorder, the biochemical and genetic defects that lead to hypertriglyceridaemia have been well characterized. These individuals present with the familial chylomicronaemia syndrome, a rare genetic disorder that is inherited as an autosomal recessive trait, and is characterized by severe fasting hypertriglyceridaemia, massive accumulations of chylomicrons in plasma, and recurrent bouts of pancreatitis. The two major causes of the familial chylomicronaemia syndrome are a deficiency of the enzyme, lipoprotein lipase (LPL), or its cofactor, apolipoprotein (apo) C-II. Together, these two proteins initiate the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins. In the past decade our understanding of the underlying molecular defects that lead to familial chylomicronaemia has been greatly enhanced by the identification of mutations in the genes for LPL and apoC-II. Characterization of these defects has provided new insights into the structure and function of apoC-II and LPL and established the important role that these two proteins play in normal triglyceride metabolism.