Autosomal recessive hypercholesterolaemia: discrimination of ARH protein and LDLR function in the homozygous FH phenotype

BACKGROUND: Phenocopies of homozygous familial hypercholesterolemia (hoFH) having autosomal recessive inheritance, were recently found to arise from defects in the LDL receptor (LDLR) adapter protein, called ARH, which facilitates the clearance of circulating LDL. Discrimination between the two causes of the phenotype at a clinical level may not be possible when parents display moderate hypercholesterolaemia. An effective strategy is thus required to identify the appropriate mechanism for the disorder. METHODS: Fibroblast LDL uptake studies were coupled with Western blotting for ARH protein in cell extracts, to identify the defective gene before DNA studies were initiated. Two subjects with the hoFH phenotype, but with indeterminate dyslipidaemia in their parents, were fully worked up. RESULTS: Defective LDL metabolism was established in both patients by functional and protein studies and further confirmed by detecting deleterious mutations, in the LDLR and ARH genes. The ARH patient is the first subject of Negroid identity to be described and records a specific mutation in this racial grouping. CONCLUSION: This study highlights the occasional complexity and uncertainty of a clinical diagnosis of hoFH and presents Western blotting of leucocyte extracts for ARH protein, as a rapid strategy for the detection of ARH before sequencing the gene for mutation(s). This strategy may be particularly useful in populations where founder mutations for ARH and LDLR defects are rare or co-exist.     

Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia

Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS((R)) analysis of binding and uptake of fluorescent LDL or anti-LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.     

Disruption of LDL but not VLDL clearance in autosomal recessive hypercholesterolemia

Genetic defects in LDL clearance result in severe hypercholesterolemia and premature atherosclerosis. Mutations in the LDL receptor (LDLR) cause familial hypercholesterolemia (FH), the most severe form of genetic hypercholesterolemia. A phenocopy of FH, autosomal recessive hypercholesterolemia (ARH), is due to mutations in an adaptor protein involved in LDLR internalization. Despite comparable reductions in LDL clearance rates, plasma LDL levels are substantially lower in ARH than in FH. To determine the metabolic basis for this difference, we examined the synthesis and catabolism of VLDL in murine models of FH (Ldlr(-/-)) and ARH (Arh(-/-)). The hyperlipidemic response to a high-sucrose diet was greatly attenuated in Arh(-/-) mice compared with Ldlr(-/-) mice despite similar rates of VLDL secretion. The rate of VLDL clearance was significantly higher in Arh(-/-) mice than in Ldlr(-/-) mice, suggesting that LDLR-dependent uptake of VLDL is maintained in the absence of ARH. Consistent with these findings, hepatocytes from Arh(-/-) mice (but not Ldlr(-/-) mice) internalized beta-migrating VLDL (beta-VLDL). These results demonstrate that ARH is not required for LDLR-dependent uptake of VLDL by the liver. The preservation of VLDL remnant clearance attenuates the phenotype of ARH and likely contributes to greater responsiveness to statins in ARH compared with FH.     

Low-density lipoprotein receptor activity in Epstein-Barr virus-transformed lymphocytes from heterozygotes for the D374Y mutation in the PCSK9 gene

OBJECTIVE: Missense mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have been found to cause autosomal dominant hypercholesterolemia. The objective of this study was to investigate possible mechanisms by which mutation D374Y in the PCSK9 gene causes hypercholesterolemia. MATERIAL AND METHODS: Binding and internalization of low-density lipoprotein LDL in Epstein-Barr virus (EBV)-transformed lymphocytes from D374Y heterozygotes were examined. The autocatalytic activity of the D374Y mutant was studied in transiently transfected HEK293 cells. RESULTS: As determined by Western blot analysis of transiently transfected HEK293 cells, the autocatalytic activity of the D374Y mutant was approximately 95% of the wild-type. Levels of PCSK9 mRNA in EBV-transformed lymphocytes from D374Y heterozygotes and normal controls were similar and less than 1/1000 of the level in HepG2 cells. The amount of cell surface LDL receptors (LDLRs) in EBV-transformed lymphocytes from five D374Y heterozygotes was non-significantly increased by 17% compared with the amount in normal controls. LDLR-dependent binding and internalization of LDL in EBV-transformed lymphocytes from D374Y heterozygotes were non-significantly reduced by 11% and 12%, respectively, compared to the corresponding values in normal controls. CONCLUSIONS: LDLR-mediated endocytosis of LDL is not reduced in EBV-transformed lymphocytes from D374Y heterozygotes. Because of the extremely low levels of PCSK9 mRNA in EBV-transformed lymphocytes, it is possible that the LDLR-dependent endocytosis of LDL could be more severely affected in hepatocytes from D374Y heterozygotes than in EBV-transformed lymphocytes.     

Primary hypercholesterolemia: genetic causes and treatment of five monogenic disorders

Coronary heart disease is a major cause of death in Europe and the USA. Insudation of atherogenic lipoproteins, including low-density lipoprotein (LDL), into the artery wall is integral to atherosclerosis. It is clear that numerous genetic loci contribute to increased plasma levels of LDL. However, five specific monogenic disorders, three of which have been reported recently, are known to increase LDL. These are familial hypercholesterolemia (LDL receptor gene: LDLR); familial ligand-defective apoB- 100 (apoB gene: APOB); autosomal recessive hypercholesterolemia (ARH gene); sitosterolemia (ABCG5 or ABCG8 genes) and cholesterol 7alpha-hydroxylase deficiency (CYP7A1 gene). This review relates the mechanisms underlying these five disorders with specific therapeutic interventions.     

Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing

Unraveling the mechanisms controlling remnant lipoprotein clearance is important, as these lipoproteins are highly atherogenic. The most critical molecule in this process is apoE, which mediates high-affinity binding of remnant lipoproteins to members of the LDL receptor (LDLR) family and cell-surface heparan sulfate proteoglycans (HSPGs), which have been shown to play major independent as well as cooperative roles in remnant lipoprotein clearance. While all the players may have been identified, our understanding of how they interact and function together continues to evolve. In this issue of the JCI, MacArthur et al. (see the related article beginning on page 153) demonstrated that HSPGs under normal physiological conditions are critically important in the clearance of remnant lipoproteins, independent of LDLR family members. The complexity of VLDL and chylomicron remnant clearance was exemplified by the studies of Jones et al., also in this issue (see the related article beginning on page 165). Despite defective clearance of LDL in mice with a deficiency in the adaptor protein controlling internalization of the LDLR, called autosomal recessive hypercholesterolemia (ARH), remnant lipoprotein clearance was not grossly abnormal. A likely explanation is that the abnormal LDLRs bind the remnants and then transfer them to another acceptor for internalization. While the studies clearly demonstrate that the LDLR-related protein 1 is not involved and suggest a role for an additional unidentified receptor, it remains a possibility that HSPGs are responsible for remnant uptake by hepatocytes in the presence of defective LDLR internalization.     

Primary hypercholesterolemia: genetic causes and treatment of five monogenic disorders

Coronary heart disease is a major cause of death in Europe and the USA. Insudation of atherogenic lipoproteins, including low-density lipoprotein (LDL), into the artery wall is integral to atherosclerosis. It is clear that numerous genetic loci contribute to increased plasma levels of LDL. However, five specific monogenic disorders, three of which have been reported recently, are known to increase LDL. These are familial hypercholesterolemia (LDL receptor gene: LDLR); familial ligand-defective apoB100 (apoB gene: APOB); autosomal recessive hypercholesterolemia (ARH gene); sitosterolemia (ABCG5 or ABCG8 genes) and cholesterol 7αhydroxylase deficiency (CYP7A1 gene). This review relates the mechanisms underlying these five disorders with specific therapeutic interventions.     

ARH missense polymorphisms and plasma cholesterol levels.

Mutations in a putative low-density lipoprotein (LDL) receptor adaptor protein called ARH have been recently described in patients with autosomal recessive hypercholesterolemia (ARH). ARH plays a tissue-specific role in determination of LDL receptor function. In the ARH gene three mismatched polymorphisms have been detected: Pro202Ser, Pro202His and Arg238Trp. These are of putative interest in plasma cholesterol level determination. To evaluate the effect of polymorphisms on plasma cholesterol levels, all polymorphisms were analyzed by PCR and restriction enzyme analysis by MnII, HpyCH4IV and SacII in 100 Caucasian males with high (>90%, 6.29 +/- 0.89 mmol/l), and 100 males with low (<10%, 3.60 +/- 0.57 mmol/l), total plasma cholesterol levels. No significant differences were observed in frequencies of ARH genotypes or alleles between these two extreme groups. These results suggest that ARH polymorphisms are unlikely to be important genetic determinants of plasma cholesterol levels.     

Familial defective apo B-100, characterization of an Italian family

Abstract. Familial defective apolipoprotein (apo) B-100 is a genetic disorder presenting with hypercholes-terolaemia and abnormal low-density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B-100 gene. In the present study thirteen members of a family with moderate hypercholesterolaemia (250–350 mg dl^-1) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolaemia (FH, receptor deficiency). We then studied the interaction between LDL and their receptors by an in vitro cell binding assay. LDL from nine affected members displayed a reduced affinity (2·5-fold) for the receptor, and were less effective than LDL from control and unaffected members in suppressing LDL receptor expression and in stimulating cholesterol esterification. LDL from the affected members had normal electrophoretic mobility, size and chemical composition. Partial delipidation did not modify the LDL binding defect. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolaemics. Analysis of DNA from family members showed a point mutation leading to an Arg to Gln substitution at amino acid 3500 of the mature protein that segregated with hypercholesterolaemia and LDL defective binding. We conclude that this family is affected by familial defective apolipoprotein B-100 (FDB).     

Genetics of the low density lipoprotein receptor. Diminished receptor activity in lymphocytes from heterozygotes with familial hypercholesterolemia.

Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.     

Two common low density lipoprotein receptor gene mutations cause familial hypercholesterolemia in Afrikaners.

Familial hypercholesterolemia (FH), an autosomal dominant disease caused by mutations in the LDL receptor gene, is five times more frequent in the Afrikaner population of South Africa than it is in the population of the United States and Europe. It has been proposed that the high frequency is due to a founder effect. In this paper, we characterized 24 mutant LDL receptor alleles from 12 Afrikaner individuals homozygous for FH. We identified two mutations that together makeup greater than 95% of the mutant LDL receptor genes represented in our sample. Both mutations were basepair substitutions that result in single-amino acid changes. Each mutation can be detected readily with the polymerase chain reaction and restriction analysis. The finding of two common LDL receptor mutations in the Afrikaner FH homozygotes predicts that these mutations will predominate in the Afrikaner population and that the high frequency of FH is due to a founder effect. The increased incidence of ischemic heart disease in the Afrikaner population may in part be due to the high frequency of these two mutations in the LDL receptor gene.     

Identification and characterization of novel low-density lipoprotein receptor mutations of familial hypercholesterolaemia patients in Taiwan

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal dominant disease associated with a very high risk of coronary vascular disease. The study objective was to identify patients with FH in Taiwan and characterize novel mutations. MATERIALS AND METHODS: Fifty-one patients with suspected FH living in Taiwan were screened for mutations in both the low-density lipoprotein (LDL) receptor and the apolipoprotein (apoB) genes using the multiplex polymerase chain reaction and exon-by-exon DNA sequencing technique. Functional consequences on LDL receptor activity were characterized in vitro for novel mutations and family pedigree was also analyzed. RESULTS: Thirteen different functional mutations in the LDL receptor gene and one mutation in the apoB gene were found in 21 patients. Among the 13 mutations in the LDL receptor gene, 10 were single-point missense mutations, one was a two-point mutation in the same allele, one was a non-sense mutation and one was a frame-shift mutation. There were three novel mutations, including two missense mutations (M510K and W512R) and one frame-shift mutation (1953 delTA mutation). The characterization of missense M510K retained 36.2% of the activity of the normal receptor. Conversely, frame-shift 1953 delTA and missense W512R led to defective proteins, with only 0-6% of normal receptor activity. CONCLUSIONS: The study identified 13 LDL receptor gene mutations and characterized three novel mutations causing FH in Taiwan. This facilitated a better understanding of FH among the Chinese population and may enable diagnosis of FH at the molecular level at a presymptomatic, early age.     

Microsomal transfer protein (MTP) inhibition—a novel approach to the treatment of homozygous hypercholesterolemia

Abstract

Homozygous familial hypercholesterolemia (HoFH) represents the most severe lipoprotein disorder, generally attributable to mutation(s) of the low-density lipoprotein receptor (LDL-R), i.e. autosomal dominant hypercholesterolemia type 1 (ADH1). Much lower percentages are due to alterations of apolipoprotein B (ADH2), or gain-of-function mutations of proprotein convertase subtilisin/kexin type 9 (PCSK9) (ADH3). In certain geographical areas a significant number of patients may be affected by an autosomal recessive hypercholesterolemia (ARH). Mutations may be also combined (two mutations of the same gene, compound heterozygosity), or two in different genes (double heterozygosity). Among the most innovative therapeutic approaches made available recently, inhibitors of the microsomal transfer protein (MTP) system have shown a high clinical potential. MTP plays a critical role in the assembly/secretion of very-low-density lipoproteins (VLDL), and its absence leads to apo B deficiency. MTP antagonists dramatically lower LDL-cholesterol (LDL-C) in animals, although a reported increase of liver fat delayed their clinical development. Lomitapide, the best-studied MTP inhibitor, reduces LDL-C by 50% or more in HoFH patients, with modest, reversible, liver steatosis. Recent US approval has confirmed an acceptable tolerability, provided patients adhere to a strictly low-fat regimen. There are no clinical data on atherosclerosis reduction/regression, but animal models provide encouraging results.     

Low density lipoprotein receptors: preliminary results on "in vivo" study

Plasmatic levels of low density lipoproteins (LDL) are regulated by the receptor pathway and most LDL receptor are located in the liver. A receptor defect due to genetic mutations of the LDL receptor gene is the cause of familial hypercholesterolemia (F. H.), a disease characterized by high cholesterol levels and premature atherosclerosis. Injection of autologous radiolabelled LDL, followed by hepatic scintiscanning, can be used to obtain "in vivo" quantification of hepatic receptor activity, both in normal and hypercholesterolemic patients. In this study we observed no hepatic increase of radioactivity in patients affected by F. H., confirming the liver receptor defect. Scintigraphy is a non-invasive technique which can be used to diagnose this disease and to monitor the efficacy of hypolipidemic therapy.     

Familial hypercholesterolemia: epidemiology, Neolithic origins and modern geographic distribution

The elucidation of the molecular basis of familial hypercholesterolemia (FH) by Brown and Goldstein about three decades ago provided the most convincing evidence for a causative relationship between a high plasma level of low-density lipoprotein (LDL) cholesterol and the conditions of atherosclerosis and premature atherosclerotic cardiovascular disease. Today, with a prevalence of about one in 500 individuals, FH remains the most common monogenic disorder of lipoprotein metabolism, and is mainly due to mutations in the LDL receptor (LDLR) gene that lead to the plasma accumulation of cholesterol ester-laden LDL particles. Another form of autosomal dominant hypercholesterolemia, familial defective apolipoprotein B-100, a genocopy of FH caused by defects in the APOB gene that lead to decreased clearance of LDL, is now established as a significant cause of coronary heart disease. Yet another form, due to mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, has been recently identified that similarly causes decreased clearance of LDL by novel mechanisms also involving the hepatic LDLR endocytotic pathway. Recent advances in molecular genotyping technology have yielded a staggering amount of detail about human genetic diversity at the single nucleotide level in both private and public databases including the International HapMap Consortium. This, as well as the availability of ancient human DNA from burial sites and the development of new statistical methods, now provide an unprecedented capacity to study human demography and the ability to examine the genealogical ties between ancient and modern people. The aim of this article is to review the epidemiology of FH, and to attempt to draw inferences from our knowledge at a DNA level of inherited hypercholesterolemia of contemporary people that may contribute to the understanding of human population history and adaptation that resulted in the massive demographic expansion following the adoption of agriculture in the Neolithic period.     

Finnish type of low density lipoprotein receptor gene mutation (FH-Helsinki) deletes exons encoding the carboxy-terminal part of the receptor and creates an internalization-defective phenotype.

A specific type of gene mutation affecting the LDL receptor has been found in many Finnish patients with familial hypercholesterolemia (FH). The mutant allele is characterized by a 9.5-kb deletion extending from intron 15 to exon 18. Molecular cloning and sequencing of a cDNA segment corresponding to the deleted allele indicated that the mutant receptor differs radically from the normal one because of loss of the domains encoded by exons 16, 17, and 18. The carboxy-terminal portion of the normal receptor, comprising the amino acids 750-839, has been replaced by an unrelated stretch of 55 amino acids. The mutant allele was found to occur in 23 (50%) of 46 unrelated FH patients with an established functional defect in the LDL receptor. In cultured fibroblasts from the FH patients with the 9.5-kb deletion, both receptor-mediated binding and internalization of 125I-LDL were lower than normal, the former, on average, by 25%, and the latter, on average, by 50%. This combined functional defect probably results from both impaired attachment and impaired internalization of the mutated receptor. It remains to be investigated whether this Finnish type of LDL receptor gene mutation, here designated FH-Helsinki, occurs in other ethnic groups.     

Familial Hypercholesterolemia: EVIDENCE FOR A NEWLY RECOGNIZED MUTATION DETERMINING INCREASED FIBROBLAST RECEPTOR AFFINITY BUT DECREASED CAPACITY FOR LOW DENSITY LIPOPROTEIN IN TWO SIBLINGS

Cultured skin fibroblasts were obtained from two siblings with classic clinical features of homozygous familial hypercholesterolemia. Plasma cholesterol values were 970 and 802 mg/100 ml in the siblings, 332 mg/100 ml in the mother, and 426 mg/100 ml in the father. Fibroblast receptor-specific capacity for binding and degradation of (125)I-low density lipoprotein (LDL) at 37 degrees C was 11% of normal, consistent with the diagnosis of "homozygous LDL receptor-defective" hypercholesterolemia, a disorder in which LDL binding activity is low but detectable.The residual LDL receptor activity was clearly qualitatively abnormal. The Michaelis constant (K(m)) for (125)I-LDL was reduced to 20-40% of normal, indicating a substantially increased affinity for LDL. Increased affinity and reduced capacity for (125)I-LDL are also found when normal fibroblasts are assayed at 4 degrees C. As the temperature is raised to 37 degrees C surface LDL binding affinity decreases while capacity increases. At 4 degrees C the fibroblasts of our subjects had an affinity for LDL indistinguishable from normal cells assayed at that temperature and a binding capacity 23% of normal. However, only small changes in affinity and capacity occurred upon increasing the temperature to 37 degrees C. When (125)I-apoprotein E-phospholipid vesicles were bound at 37 degrees C the receptor deficiency appeared only half as severe as when (125)I-LDL was used as ligand.A family study suggests that the siblings are genetic compounds rather than homozygotes, having inherited a mutant maternal gene causing absent or silent LDL receptors and a mutant paternal gene resulting in qualitatively altered LDL receptors. It is not clear whether these defects are present at the same or different genetic loci. The altered receptors are characterized by increased affinity and moderately reduced capacity for LDL at 37 degrees C and are accompanied by hypercholesterolemia at least as severe as that associated with familial hypercholesterolemia with absent or nonfunctional LDL receptors.     

Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors.

This paper describes an unusual kindred with familial hypercholesterolemia in which one-third of the relatives with a mutant LDL receptor gene have normal plasma cholesterol concentrations. The proband, a 9-yr-old boy with a plasma cholesterol value greater than 500 mg/dl, is homozygous for a point mutation that changes Ser156 to Leu in the LDL receptor. This substitution in the fourth repeat of the ligand binding domain slows the transport of the protein to the cell surface. The defective receptor cannot bind LDL, which contains apo B-100, but it does bind beta-migrating VLDL, which contains apo E in addition to apo B-100. Although the mother is heterozygous for this mutation, her LDL-cholesterol concentration is consistently in the 28th percentile for the population. Through direct examination of genomic DNA, we identified the mutant gene in heterozygous form in 17 of the mother's relatives, five of whom had normal LDL-cholesterol values. The pedigree was consistent with dominant transmission of a single gene that ameliorates or suppresses the hypercholesterolemic effect of the LDL receptor mutation. Through linkage analysis, we excluded the possibility that this suppressor gene was an allele at the LDL receptor locus. We also excluded the genes for the two ligands for the LDL receptor, apo B-100 and apo E. The existence of this putative suppressor gene may explain the occasional observation of normal LDL-cholesterol concentrations in heterozygotes for LDL receptor mutations.