Estimation of the prevalence of cholesteryl ester storage disorder in a cohort of patients with clinical features of familial hypercholesterolaemia.

BACKGROUND AND AIM: Familial hypercholesterolaemia is caused by variants in the low-density lipoprotein cholesterol metabolic pathway involving LDLR, APOB and PCSK9 genes. A national genetic testing service in Wales, UK has observed that no familial hypercholesterolaemia variant is found in almost 80% patients with the familial hypercholesterolaemia phenotype. It has recently been suggested that some adult patients with a familial hypercholesterolaemia phenotype may have cholesteryl ester storage disease which can also present as a mixed hyperlipidaemia. The commonest genetic cause of cholesteryl ester storage disease is an exon 8 splice junction variant in the LIPA gene (rs116928232, c.894G>A; E8SJM) previously found to have an allele frequency of 0.0011 (1 in 450 individuals) in a large European population. This study investigated the prevalence of the E8SJM in patients with a familial hypercholesterolaemia phenotype in Wales, UK. METHOD: A total of 1203 patients with a clinical suspicion of familial hypercholesterolaemia but no familial hypercholesterolaemia variant were invited to participate. Of these, 668 patients provided informed written consent. Stored DNA samples from 663 patients were genotyped for the E8SJM variant. RESULTS: Three heterozygotes were identified (allele frequency 0.0023). Whole gene sequencing of the LIPA gene was undertaken in these three individuals, but no other variants were found. Therefore, there were no cholesteryl ester storage disease patients (homozygote or compound heterozygote) identified in this cohort. CONCLUSION: The allele frequency 0.0023 (1 in 221 individuals) for the E8SJM variant was more prevalent in this cohort than in a European population study; however, no cholesteryl ester storage disease homozygotes were identified. We found no evidence to support routine testing for cholesteryl ester storage disease in adult patients with a familial hypercholesterolaemia phenotype.

Cigarette smokers differ in their handling of natural (RRR) and synthetic (all rac) alpha-tocopherol: a biokinetic study in apoE4 male subjects.

We have compared the biokinetics of deuterated natural (RRR) and synthetic (all rac) alpha-tocopherol in male apoE4-carrying smokers and nonsmokers. In a randomized, crossover study subjects underwent two 4-week treatments (400 mg/day) with undeuterated RRR- and all rac-alpha-tocopheryl acetate around a 12-week washout. Before and after each supplementation period subjects underwent a biokinetic protocol (48 h) with 150 mg deuterated RRR- or all rac-alpha-tocopheryl acetate. During the biokinetic protocols, the elimination of endogenous plasma alpha-tocopherol was significantly faster in smokers (P < 0.05). However, smokers had a lower uptake of deuterated RRR than nonsmokers, but there was no difference in uptake of deuterated all rac. The supplementation regimes significantly raised plasma alpha-tocopherol (P < 0.001) with no differences in response between smokers and nonsmokers or between alpha-tocopherol forms. Smokers had significantly lower excretion of alpha-carboxyethyl-hydroxychroman than nonsmokers following supplementation (P < 0.05). Nonsmokers excreted more alpha-carboxyethyl-hydroxychroman following RRR than all rac; however, smokers did not differ in excretion between forms. At baseline, smokers had significantly lower ascorbate (P < 0.01) and higher F(2)-isoprostanes (P < 0.05). F(2)-isoprostanes in smokers remained unchanged during the study, but increased in nonsmokers following alpha-tocopherol supplementation. These data suggest that apoE4-carrying smokers and nonsmokers differ in their handling of natural and synthetic alpha-tocopherol.

Progression of severe atherosclerosis and increased arterial pulse pressure in the newly developed heritable mixed hyperlipidaemic rabbits.

We have recently segregated a new line of rabbit, named TGH, with severely high levels of plasma triglyceride and cholesterol. The aim of the present study was to investigate the progression of atherosclerosis and haemodynamic parameters in TGH rabbits. 2. Japanese white (JW) and TGH rabbits (24-27 months old) were anaesthetized with ketamine and xylazine. Plasma concentrations of triglyceride were 63.1 8.0 and 446.0 35.2 mg/dL in JW and TGH rabbits, respectively. Blood pressure was measured by a catheter implanted in the femoral artery. Histological examinations were performed using haematoxylin-eosin and elastica-Masson trichrome staining to detect atherosclerotic lesions. 3. The JW rabbits had no atherosclerotic lesions. In TGH rabbits, severe atherosclerotic lesions were observed throughout the aorta, especially in the aortic arch. Basal femoral arterial pressure was not significantly different between JW and TGH rabbits. However, the basal pulse pressure in TGH rabbits (48.3 4.5 mmHg) was significantly greater than that of JW rabbits (28.0 5.6 mmHg). Intravenous infusion of N(G)-nitro-L-arginine methyl ester (L-NAME; 26.9 mg/kg) increased the blood pressure of TGH and JW rabbits. There was no significant difference in the response to L-NAME between the two rabbit strains. 4. The present study shows that severe atherosclerotic changes develop in TGH rabbits and suggests that the hyperlipidaemia combined with hypercholesterolaemia and hypertriglyceridaemia is an important factor for promoting atherosclerosis in TGH rabbits. The greater pulse pressure in TGH rabbits may be due to the increased vascular stiffness with atherosclerosis. 5. This newly developed TGH rabbit line of heritable hypertriglyceridaemia with hypercholesterolaemia will become a useful animal model for studies on the role of hyperlipidaemia in the progression of atherosclerosis and in many atherosclerosis-related diseases.

Two cases with transient lipoprotein lipase (LPL) activity impairment: evidence for the possible involvement of an LPL inhibitor.

UNLABELLED: Two independent severe hypertriglyceridemic infants with transiently impaired lipoprotein lipase (LPL) activity were observed and the causes were explored. Both infants were female, born prematurely with low birth weight and developed hypertriglyceridemia (Fredrickson type V hyperlipidemia: high VLDL and low LDL/HDL) a few months after birth. While mass levels of their post-heparin plasma LPL and apoprotein C-II (apo C-II), a physiological activator of LPL, were normal, their post-heparin plasma LPL activities were remarkably impaired. Both of their mothers' post-heparin plasma LPL activities were slightly or moderately impaired as well, without a decrease in the LPL mass level. No mutations in the genes for LPL and apo C-II were detected in either patient. In an in vitro study with their serum at onset, we could not detect any distinct circulating inhibitors for LPL. There was no data supporting infection or autoimmune diseases, which might have an impact on LPL activity, during the follow-up period. Levels of their plasma triglyceride (TG) and total cholesterol (TC) were decreased quickly by a dietary intervention with medium-chain triglyceride (MCT) milk and kept normal even after stopping the intervention at around age 1 year. However, their low post-heparin LPL activity persisted and returned to normal at around age 2 years. Their low HDL cholesterol levels persisted even after recovery of the TG and TC levels, although lecithin:cholesterol acyltransferase (LCAT) and cholesterol-ester-transfer protein (CETP), two key enzymes of HDL metabolism, were normal throughout the course. The exact reasons why their post-heparin LPL activities were impaired for a certain period and why their HDL cholesterol levels have remained low are still unclear. CONCLUSION: Transiently impaired LPL activity with no defect in LPL enzyme induced severe hypertriglyceridemia in infants. The transient occurrence of inhibitor(s) for LPL was proposed.

Dietary fat clearance in type V hyperlipoproteinaemia secondary to a rare variant of human apolipoprotein E: the apolipoprotein E3 (Arg 136-->Ser)

This present case report describes two siblings with severe type V hyperlipoproteinaemia, diagnosed very early in life and due to the combination of the common apolipoprotein (Apo) E2 allele and rare mutant variant of ApoE, ApoE3 (Arg 136-->Ser). Phenotyping of ApoE falsely identified E2/E2 phenotype. The presence of mutated ApoE was suspected on an unusual restriction polymorphism of a Hha 1 restriction site and confirmed by sequence analysis of the cloned polymerase chain reaction fragment of exon 4 and familial segregation study. The severity of the hypertriacylglycerolaemia was modulated by the lipid content of the diet. A low-fat diet enriched in medium-chain triacylglycerol (TAG) decreased but did not normalize plasma TAG levels in both affected patients of the pedigree. A standardized lipid-enriched test meal showed a marked impairment of TAG-rich lipoprotein (TRL) clearance, especially the exogeneous TRL bearing ApoB-48 which still represented 79% of total TRL 7 h after the fat load. Finally, differences between the male and female siblings with the existence of a consanguine relationship in their parents suggested the involvement of other genetic factors in modulating the severity of phenotypic expression. This observation reinforces the usefulness of genotyping of ApoE for the characterization of genetic hypertriacylglycerolaemia and selection of the appropriate diet and treatment.

Efficacy and safety of a combination of fluvastatin and bezafibrate in patients with mixed hyperlipidaemia (FACT study).

Preliminary data suggest that fluvastatin may be safely combined with fibrates. The Fluvastatin Alone and in Combination Treatment Study examined the effects on plasma lipids and safety of a combination of fluvastatin and bezafibrate in patients with coronary artery disease and mixed hyperlipidaemia. A total of 333 patients were randomly allocated in this multicentre double-blind trial to receive 40 mg fluvastatin alone (n=80), 400 mg bezafibrate (n=86), 20 mg fluvastatin+400 mg bezafibrate (n=85) or 40 mg fluvastatin+400 mg bezafibrate (n=82) for 24 weeks. Low-density lipoprotein (LDL)-cholesterol decreased >20% in all fluvastatin-containing regimens, with significantly greater decreases compared with bezafibrate alone (P<0.001). Bezafibrate alone and fluvastatin+bezafibrate combinations resulted in greater increases in high-density lipoprotein (HDL)-cholesterol and decreases in triglycerides compared with fluvastatin alone (P<0.001). Fluvastatin (40 mg)+bezafibrate was the most effective for all lipid parameters with a decrease from baseline at endpoint in LDL-cholesterol of 24%, a decrease in triglycerides of 38% and an increase in HDL-cholesterol of 22%. All treatments were well tolerated with no increase in adverse events for combination therapy versus monotherapy, or between combination regimens. No clinically relevant liver (aspartate aminotransferase [ASAT] or alanine aminotransferase [ALAT]) greater than three times the upper limit of normal) or muscular (creatine phosphokinase (CPK) greater than four times the upper limit of normal) laboratory abnormalities were reported. This large study shows 40 mg fluvastatin in combination with 400 mg bezafibrate to be highly effective and superior to either drug given as monotherapy in mixed hyperlipidaemia, and to be safe and well tolerated.

Diabetic lipemia with maturity-onset diabetes of the young.

A 19-year-old woman with diabetic lipemia and maturity-onset diabetes of the young (MODY) is reported. Though her insulin secretory activity was preserved, she fell into mild diabetic ketoacidosis (DKA) and showed type V hyperlipidemia. Post-heparin plasma activity of lipoprotein lipase (LPL) was decreased even 10 days after initiating insulin injection but not deficient. The abnormalities in lipid metabolism were improved by long-term insulin treatment. Though the contribution of the genetic background to the lipid abnormalities is not clear, the characteristics of MODY in this patient including insulin secretory capacity under stress conditions such as DKA might play a role in the development of diabetic lipemia.

Dyslipidaemia in a boy with recurrent abdominal pain, hypersalivation and decreased lipoprotein lipase activity.

UNLABELLED: An 8-year-old boy with frequently recurring pancreatitis-like abdominal pain, Fredrickson type V dyslipidaemia, and significantly decreased post-heparin plasma lipoprotein lipase (LPL) activity is described. In order to exclude familial LPL deficiency, the complete LPL coding gene sequence was analysed revealing compound heterozygosity for two mutations (Asp9Asn, Ser447Ter) which are not supposed to considerably impair lipolytic enzyme activity. However, until now the combination of both these mutations in one patient has not been observed. In addition to the common symptoms of LPL deficiency, a striking feature of unknown origin was hypersalivation. Treatment including a fat-restricted diet, omega-3 fatty acids, and nicotinic acid led to long symptoms-free intervals. Symptoms recurred however when the diet was not strictly adhered to. CONCLUSION: LPL deficiency is a rare cause of abdominal pain in childhood and deserves careful treatment in order to avoid pancreatitis. The presented patients is a unique compound heterozygote for two mutations which do not abolish lipolytic activity in the homozygote state. Identification of other individuals with this genotype is necessary to understand the phenotype in our patient.

Familial type V hyperlipoproteinemia with hyper-remnant-like particle-cholesterol accompanied with apolipoprotein E3/E4 phenotype.

A 41-year-old woman and her 2 sons had Type V hyperlipoproteinemia. Interestingly, the patient and her younger son had higher levels of remnant-like particle (RLP)-cholesterol than her elder son; the former two had apolipoprotein E phenotype E3/E4, while the latter had E3/E3. Hyper-RLP-ch levels may be associated with the apolipoprotein E4 phenotype. Moreover, after administration of bezafibrate, the patient's triglyceride, RLP-cholesterol and RLP-triglyceride levels decreased markedly and the chylomicron fraction disappeared. Further studies may be necessary to determine if hypertriglyceridemic patients with or without apolipoprotein E4 show a greater reduction in serum TG levels with lipid-lowering agents.

Interaction between variant apolipoproteins C-II and E that affects plasma lipoprotein concentrations.

The genes for apolipoprotein (apo) C-II, a cofactor for activation of lipoprotein lipase, and apo E, a ligand for receptor-mediated uptake of triglyceride-rich lipoproteins, are physically linked on chromosome 19q13.1. In a large Caribbean Caucasian family, several individuals had clinical features of the complete absence of lipoprotein lipase activity and were homozygous for a DNA frameshift mutation of apo C-II, imparting functional inactivity to the mutant protein. Plasma from heterozygous carriers of this mutation, when compared with plasma from relatives who were noncarriers, had significantly diminished capacity to activate lipoprotein lipase in vitro. We also observed in heterozygotes for this mutation a wide range of serum lipid and lipoprotein levels. When age and sex were taken into account, the presence of a single apo E allele encoding the E4 isoform occurring in individuals with a single mutant apo C-II allele was strongly associated with higher levels of cholesterol, triglycerides, very low density lipoprotein cholesterol, and non-high density lipoprotein cholesterol when compared with those of relatives who carried neither or only one variant allele. This suggests that a single genetic mutation that usually has a recessive effect on lipoprotein metabolism can have an interactive effect on lipid phenotype when it is coinherited with a single mutation at another gene whose product affects the same metabolic pathway.

Apolipoprotein E2-Dunedin (228 Arg replaced by Cys): an apolipoprotein E2 variant with normal receptor-binding activity.

Homozygosity for the apolipoprotein (apo) E variant apoE2(158 Arg----Cys) invariably gives rise to dysbetalipoproteinemia, and when associated with obesity or a gene for hyperlipidemia, results in type III hyperlipoproteinemia. The association of the E2/2 phenotype with type IV/V hyperlipoproteinemia rather than type III hyperlipoproteinemia in identical twin brothers led us to investigate the primary structure of their apoE. Lipoprotein electrophoresis on agarose gels confirmed the presence of increased very low density lipoproteins (VLDL) and chylomicrons but little, if any, beta-VLDL, indicating that these subjects did not have dysbetalipoproteinemia. When the apoE from these twins was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a system that can distinguish apoE2(158 Arg----Cys) from all other known apoE variants, it gave rise to two components. One had the unique mobility of apoE2(158 Arg----Cys), and one migrated in the position of the other variants of apoE (and normal apoE3), indicating that the brothers were heterozygous for apoE2(158 Arg----Cys) and a second apoE2 isoform. Cysteamine modification and isoelectric focusing showed that, like apoE2(158 Arg----Cys), the second apoE2 isoform also contained two cysteine residues. The structural mutation in the second apoE2 isoform was determined by peptide sequencing. Like normal apoE3, this variant had arginine at position 158, but differed from apoE3 by the substitution of cysteine for arginine at position 228. Total apoE isolated from the brothers had the same receptor-binding activity in a competitive binding assay as a 1:1 mixture of normal apoE3 and apoE2(158 Arg----Cys).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of apolipoproteins E and C in type V hyperlipoproteinemia.

Type V hyperlipoproteinemia is characterized by elevations of chylomicron (CM) and very low density lipoprotein (VLDL) triglycerides. The development of this lipid disorder involves a multitude of metabolic derangements including deficient clearance of triglycerides and/or their increased output aggravated by obesity, diabetes, alcohol intake, or use of some hormones. Some studies have suggested that the apolipoprotein E4 phenotype is involved in this dyslipoproteinemia but this concept is still a matter of controversy. Therefore, we determined the apoE phenotype in 21 patients with severe hypertriglyceridemia classified as type V. Their apoE4 gene frequency was 0.595 which is 2.6-fold higher (P less than 0.001) than that in the Finnish population. Correspondingly, their apoE3 gene frequency was lower than that in the normal population. No differences were noted in plasma lipoproteins of the apoE4 phenotypes and the other type V subjects. The apolipoprotein C-II and C-III distribution was similar to that in normolipidemic subjects. The results suggest that apoE4 may be involved in the development of type V hyperlipoproteinemia.

Abnormal in vivo metabolism of apolipoprotein E4 in humans.

Apolipoprotein E (apoE) is important in modulating the catabolism of remnants of triglyceride-rich lipoprotein particles. It is a polymorphic protein with the three common alleles coding for apoE2, apoE3, and apoE4. ApoE3 is considered the normal isoform, while apoE4 is associated both with hypercholesterolemia and type V hyperlipoproteinemia. We quantitated the kinetics of metabolism of apoE4 in 19 normolipidemic apoE3 homozygotes and 1 normolipidemic apoE4 homozygote, and compared this with the metabolism of apoE3 in 12 normolipidemic apoE3 homozygotes. In the apoE3 homozygous subjects, apoE4 was catabolized twice as fast as apoE3, with a mean plasma residence time of 0.37 +/- 0.01 d (+/- SEM) and 0.73 +/- 0.05 (P less than 0.001), respectively. When plasma was fractionated into the lipoprotein subclasses, the greatest amount of labeled apoE4 was present on very low density lipoproteins, while the largest fraction of labeled apoE3 was associated with high density lipoproteins. The plasma apoE concentration was decreased in an apoE4 homozygote compared with the apoE3 homozygotes (3.11 mg/dl vs. 4.83 +/- 0.35 mg/dl). The reduced apoE4 concentration was entirely due to a decreased apoE4 residence time in the apoE4 homozygote (0.36 d vs. 0.73 +/- 0.05 d for apoE3 in apoE3 homozygotes). These results indicate that apoE4 is kinetically different than apoE3, and suggest that the presence of apoE4 in hypercholesterolemic and type V hyperlipoproteinemic individuals may play an important pathophysiological role in the development of these dyslipoproteinemias.