Apolipoprotein B metabolism in homozygous familial hypercholesterolemia

This report describes the metabolism of apolipoprotein B-containing lipoproteins in seven familial hypercholesterolemic (FH) homozygotes and compares the results to the values obtained from five healthy control subjects. The concentration, composition, and metabolism of large, triglyceride-rich very low density lipoproteins (VLDL1, Sf 60-400) were the same in the control and FH groups, indicating that this component of the VLDL delipidation cascade ws unaffected by the absence of receptors. In contrast, familial hypercholesterolemic small VLDL2 (Sf 20-60) was enriched with cholesterol and depleted in triglyceride. Moreover, its plasma concentration was elevated as a result of an increase in its synthesis and a defect in the removal of a remnant population within this density interval. The latter accounted for up to 50% of the total mass of the fraction. Onward transfer of apolipoprotein B (apoB) from small VLDL through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL) was retarded, suggesting that receptors were involved in this supposedly lipase-mediated event. IDL and LDL concentrations increased up to fourfold above normal in the plasma of the FH patients due partly to the delay in maturation and partly to defective direct catabolism. We conclude that the LDL receptor plays multiple and important roles in the metabolism and transformation of apoB-containing particles in the Sf 0-400 flotation interval.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Clinical course of homozygous familial hypercholesterolemia during childhood: report on 4 unrelated patients with homozygous or compound heterozygous mutations in theLDLR gene

Natural history of the disease in 4 unrelated Polish children with homozygous familial hypercholesterolemia (FH) is described. Their phenotypic homozygosity was established by identification of known LDLR gene mutations on both alleles, respectively: p.G592E & p.G592E in Patient 1; p.G592E & p.C667Y in Patient 2; p.S177L & p.R350X in Patient 3; and p.G592E & deletion in the promoter region, exons 1 and 2 in Patient 4. Heterozygosity of the mutations was revealed in all patients' mothers and fathers (obligatory heterozygotes) and in 1 out of 4 siblings studied. FH was diagnosed at the age of 4 months to 9 years by cholesterol screening among family members of patients with early cardiovascular disease episodes. At the time of FH detection, the children were asymptomatic. Only in 2, some skin eruptions were found. Antihyperlipidemic therapy was started, including a lipid-lowering diet, cholestyramine, and HMG-CoA inhibitors if necessary. No cardiovascular symptoms appeared during the observation up to the age of 18, 20, 19, and 17 years, respectively. An increase in external carotid artery diameter was found in a patient at the age of 9 years, and LDL-apheresis was introduced in his therapy. We conclude that the analysis of LDLR gene mutations in the studied FH children made it possible to identify 4 presymptomatic FH homozygotes and to introduce early appropriate treatment. Multicenter analysis of such persons would finally determine if the early lipid-lowering procedures can significantly reduce the risk of premature cardiovascular disease in homozygous FH.     

Similar content of phospholipids and gangliosides in normal and homozygous familial hypercholesterolemia fibroblasts.

The cellular content of total and individual phospholipids and gangliosides was measured in fibroblasts cultured from four normal subjects, three patients with lysosomal lipid storage diseases, and two subjects with homozygous familial hypercholesterolemia. Measurements were made on cells grown in medium containing fetal calf serum under conditions in which normal cells derive cholesterol for cell growth from low density lipoprotein present in the fetal calf serum, whereas familial hypercholesterolemia homozygote cells, which lack cell surface low density lipoprotein receptors, derive cholesterol from endogenous synthesis. No difference was observed in the cellular content of total or individual phospholipids and gangliosides in the normal and familial hypercholesterolemia homozygote cells. In contrast, cells from a patient with Niemann-Pick disease and a patient with Sandhoff disease showed elevations in the content of sphingomyelin and complex gangliosides, respectively.     

Impact of LDL apheresis on atheroprotective reverse cholesterol transport pathway in familial hypercholesterolemia

In familial hypercholesterolemia (FH), low HDL cholesterol (HDL-C) levels are associated with functional alterations of HDL particles that reduce their capacity to mediate the reverse cholesterol transport (RCT) pathway. The objective of this study was to evaluate the consequences of LDL apheresis on the efficacy of the RCT pathway in FH patients. LDL apheresis markedly reduced abnormal accelerated cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer from HDL to LDL, thus reducing their CE content. Equally, we observed a major decrease (-53%; P < 0.0001) in pre-β1-HDL levels. The capacity of whole plasma to mediate free cholesterol efflux from human macrophages was reduced (-15%; P < 0.02) following LDL apheresis. Such reduction resulted from a marked decrease in the ABCA1-dependent efflux (-71%; P < 0.0001) in the scavenger receptor class B type I-dependent efflux (-21%; P < 0.0001) and in the ABCG1-dependent pathway (-15%; P < 0.04). However, HDL particles isolated from FH patients before and after LDL apheresis displayed a similar capacity to mediate cellular free cholesterol efflux or to deliver CE to hepatic cells. We demonstrate that rapid removal of circulating lipoprotein particles by LDL apheresis transitorily reduces RCT. However, LDL apheresis is without impact on the intrinsic ability of HDL particles to promote either cellular free cholesterol efflux from macrophages or to deliver CE to hepatic cells.     

Elevated serum MMP-9/TIMP-1 ratio in patients with homozygous familial hypercholesterolemia: Effects of LDL-apheresis

ObjectiveMatrix degradation within an atherosclerotic plaque is an important pathogenic factor in atherosclerosis, and is largely modulated by the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors (i.e., tissue inhibitor of MMPs [TIMPs]). Familial hypercholesterolemia (FH) is a rare inherited disorder associated with premature coronary heart disease. The aim of the present study was to examine MMP-9 and TIMP-1 on plasma and cellular mRNA levels in homozygous FH patients (n = 7) compared with age- and sex-matched heterozygous FH patients (n = 6), and with healthy subjects (n = 7), and to test whether once-weekly LDL-apheresis (three consecutive sessions) of homozygous FH patients show short-term effects on these variables.ResultsThe main findings were that (i) Compared to healthy control subjects, homozygous FH patients have significantly higher serum levels of MMP-9 and lower levels of TIMP-1, and consequently significantly higher MMP-9/TIMP-1 ratio, potentially reflecting higher MMP-9 activity. (ii) Peripheral blood mononuclear cells (PBMC) isolated from FH homozygotes have significantly higher mRNA levels of MMP-9 compared to cells from heterozygotes. (iii) TNFα-stimulated PBMC from FH homozygotes released borderline-significantly more MMP-9 than cells from heterozygotes and healthy controls. (iv) LDL-apheresis (one day before treatment versus fifteen days later, on the day after the weekly treatment) had no significant short-term effect on any of the MMP-9 and TIMP-1 variables measured in serum and cells.ConclusionsThe data may suggest that homozygous FH patients have an enhanced matrix degrading potential as compared with heterozygous FH patients and healthy controls, potentially contributing to the increased cardiovascular risk observed in these patients.     

Different genes and polymorphisms affecting high-density lipoprotein cholesterol levels in Greek familial hypercholesterolemia patients

Familial Hypercholesterolemia (FH) is a genetic disorder characterized by high low-density lipoprotein cholesterol (LDL-C) concentrations that frequently gives rise to premature coronary artery disease. The clinical expression of FH is highly variable, even in patients carrying the same LDL receptor gene mutation. This variability may be due to environmental and other genetic factors. We investigated the effect of APOCIII T1100C, FV Gln506Arg, ADRB2 Glu27Gln, SELE Ser128Arg, SELE Leu554Phe, and ENaCa Ala663Thr polymorphisms on the HDL-C variations in 84 patients with FH. For ApoCIII T1100C, subjects with the TT genotype presented higher HDL-C levels than the other genotype groups (p = 0.046). Similarly the presence of the Gln allele in ADRB2 27 Glu/Gln heterozygotes and ADRB2 27 Gln/Gln homozygotes was associated with higher HDL-C levels (p = 0.014). Among the other polymorphisms tested, none of them were associated with variations in HDL-C levels. The influence of each polymorphism on lipid concentrations was evaluated with linear regression analyses after adjustment for age and sex. Among the variables studied including total cholesterol, LDL-C, high-density lipoprotein (HDL)-C, triglycerides, apolipoprotein A (Apo-A) and B (Apo-B), and lipoprotein alpha (LP alpha), HDL-C concentration was significantly different in models applied for polymorphisms ApoCIII T1100C, FV Gln506Arg, and ADRB2 Glu27Gln (p = 0.01, p = 0.018, p = 0.04, respectively). These results suggest that HDL-C levels in FH heterozygotes may be affected by several different genetic variants.     

Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin

South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp₆₉(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu₁₁₉GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in amy of the South African Indian FH of hypercholesterolemic patients that were screened.     

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH)

Familial hypercholesterolemia (FH), Niemann-Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.     

Suppression of cholesterol synthesis in cultured fibroblasts from a patient with homozygous familial hypercholesterolemia by her own low density lipoprotein density fraction A possible role of apolipoprotein E

The suppression of cellular cholesterol synthesis by low density lipoprotein (LDL) from a normal and from a homozygous familial hypercholesterolemic subject was measured on normal fibroblasts and on fibroblasts derived from the same homozygous familial hypercholesterolemic patient. On normal fibroblasts both LDL preparations (denisty 1.019 to 1.063 g/ml) exerted a similar suppression of cellular cholesterol synthesis. With the homozygous familial hypercholesterolemic fibroblasts homozygous hypercholesterolemic LDL suppressed the cholesterol synthesis to a much greater extent than did LDL from a normal subject. Analysis of lipid and protein composition of both LDL preparations showed that homozygous hypercholesterolemic LDL differs from normal LDL. In the homozygous hypercholesterolemic LDL preparation the ratio phosphatidylcholine to sphingomyelin is decreased, and even when taking a narrower density range (1.023 to 1.045 g/ml), apolipoprotein E is present. In this homozygous hypercholesterolemic LDL preparation (density range 1.023 to 1.045 g/ml) apolipoprotein E could be present as an integral LDL protein constituent or as an apolipoprotein of an HDL_c-like lipoprotein class with a floatation density similar to that of LDL. It is suggested that the presence of apolipoprotein E in the LDL density fraction from plasma from this homozygous familial hypercholesterolemic patient could offer an additional means for suppression of cellular cholesterol synthesis of this patient.     

microRNA-30c reduces plasma cholesterol in homozygous familial hypercholesterolemic and type 2 diabetic mouse models

High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce the risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein production; however, this is associated with steatosis. We previously showed that microRNA (miR)-30c lowers diet-induced hypercholesterolemia and atherosclerosis in C57BL/6J and Apoe^−/− mice. Here, we tested the effect of miR-30c on plasma lipids, transaminases, and hepatic lipids in different mouse models. Hepatic delivery of miR-30c to chow-fed leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) hypercholesterolemic and hyperglycemic mice reduced cholesterol in total plasma and VLDL/LDL by ∼28% and ∼25%, respectively, without affecting triglyceride and glucose levels. And these mice had lower plasma transaminases and creatine kinase activities than controls. Moreover, miR-30c significantly lowered plasma cholesterol and atherosclerosis in Western diet-fed Ldlr^−/− mice with no effect on plasma triglyceride, glucose, and transaminases. In these studies, hepatic lipids were similar in control and miR-30c-injected mice. Mechanistic studies showed that miR-30c reduced hepatic microsomal triglyceride transfer protein activity and lipid synthesis. Thus miR-30c reduced plasma cholesterol in several diet-induced and diabetic hypercholesterolemic mice. We speculate that miR-30c may be beneficial in lowering plasma cholesterol in different metabolic disorders independent of the origin of hypercholesterolemia.     

Enhanced degradation of trypsin-treated low density lipoprotein by fibroblasts from a patient with homozygous familial hypercholesterolemia.

When 125I-labeled native low density lipoprotein was incubated with skin fibroblasts from a patient with homozygous familial hypercholesterolemia, the observed rate of degradation of the protein moiety was less than 5% the rate observed with normal fibroblasts, in agreement with previous studies. When the low density lipoprotein had been first treated with trypsin, with release of about 20% of the protein, its degradation by the patient's fibroblasts was markedly increased 8-20-fold. In contrast, the rate of degradation of the trypsin-treated lipoprotein by normal fibroblasts was, if anything, slightly reduced. In neither the normal cells nor the patient's cells was binding to the cell surface appreciably altered by trypsin treatment of the lipoprotein. Prior incubation with cholesterol and 7-ketocholesterol reduced binding of trypsin-treated low density lipoprotein to normal cells by 67% but did not affect its binding to the patient's cells. The results show that the structural modifications induced by trypsin do not interfere with binding of low density lipoprotein to its normal high affinity receptor nor its degradation by normal cells. However, the modified lipoprotein is much more readily internalized and degraded by cells from the patient with homozygous familial hypercholesterolemia.     

Cerebral gigantism: report on two familial cases (author's transl)

Two cases of cerebral gigantism occurring in related boys (cousins of 3rd degree) are discussed. It is difficult to argue from these cases in favour of a precise type of hereditary transmission. The hypothesis of a dominant trait with weak penetrance cannot be excluded. A genetic heterogeneity of the Sotos syndrome is very likely.     

Molecular genetics of familial hypercholesterolemia in Israel

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by a multitude of low density lipoprotein receptor (LDL-R) mutations. The purpose of the current investigation was to define the spectrum of mutations causing FH in Israel and determine their relative distribution among diverse origin groups. A total of 193 FH families were recruited in Israel, 54 of them through the MED PED (Make Early Diagnosis Prevent Early Death) FH program. Molecular analysis of the LDL-R using single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) or both has been completed in 95 index cases. This analysis resulted in the identification of 15 LDL receptor mutations, including 7 novel mutations (del 197, C308G, R385W, splice junction mutation of intron 14, del 328, del 502–505, stop 10, del 165), that were present in 49 index cases (52%). The 15 mutations are mapped to three known functional domains of the receptor (7 in the LDL-binding region, 7 in the epidermal growth factor precursor homology region and 1 in the membrane-spanning region). Screening for the identified mutations in the remaining 98 index cases enabled the molecular diagnosis of 31 additional cases. It is therefore concluded that 80 out of 193 index cases (41%) harbor 1 of the 15 mutations described here. Three mutations – del₁₉₇ (FH-Lithuania), D147H (FH-Sephardic), and stop₆₆₀ (Lebanese allele) – were found in a total of 66 index cases (34%); these may be regarded as founder mutations in the three respective origin groups. In conclusion, in Israel molecular heterogeneity at the LDL receptor gene locus reflects the ethnic distribution of its origin groups. The results of the present investigation provide valuable diagnostic tools for a subset of the Israeli patients with FH who are at high risk for atherosclerosis and its complications. Received: 1 February 1996 / Revised: 22 May 1996     

HDL abnormalities in familial hypercholesterolemia: Focus on biological functions

Although a selective strong elevation in the plasma level of low-density lipoprotein (LDL) cholesterol is the hallmark of familial hypercholesterolemia (FH), also other plasma lipoprotein and lipid subspecies are changed in these patients. Several studies in FH patients have pointed to the qualitative abnormalities of high-density lipoprotein (HDL) particles, including their triglyceride and sphingomyelin enrichment, reduced capacity to promote cholesterol efflux from macrophages, impaired anti-inflammatory and anti-oxidant activities, and reduced plasma levels of miRs regulating HDL-dependent cholesterol efflux from macrophage foam cells, typical of atherosclerotic lesions. Thus, accurate understanding of HDL functionality and its disturbances in FH may serve a better estimation of the prognosis and also provide additional clues when searching for novel therapeutic choices in this disease. In spite of such a potential promise, there has been no prior comprehensive review focusing on indices of HDL function in FH patients. In the present review, we aim to fulfill this gap by identifying measures of HDL function that are impaired in FH, and by providing a concise summary on the impact of different lipid-modifying therapies on HDL functionality in FH.