Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

Rabbit and rat C-reactive proteins bind apolipoprotein B-containing lipoproteins.

Immobilized rabbit and rat C-reactive protein (CRP) were found to selectively bind apolipoprotein B (apoB)-containing lipoproteins (low density lipoprotein, LDL and very low density lipoprotein, VLDL) from whole serum in a manner similar to that previously reported with human CRP. In acute phase human serum the CRP is in a free form, not complexed with lipoprotein or any other macromolecular ligand, and in acute phase serum from most rabbits fed on a normal diet the rabbit CRP was also free. However, in acute phase serum or heparinized plasma from hypercholesterolemic rabbits part or all of the CRP was found by gel filtration and immunoelectrophoretic techniques to be complexed with beta-VLDL, an abnormal apoB-containing plasma lipoprotein present in these animals. The presence of extent in different serum samples of CRP complexed with lipoprotein correlated closely with the serum apoB concentration. The formation of complexes between native, unaggregated rabbit CRP in solution and apoB-containing lipoproteins was readily demonstrable experimentally both with the isolated proteins and in whole serum. In all cases these interactions were calcium-dependent and inhibitable by free phosphoryl choline. The present findings extend earlier work in man and the rabbit and indicate that among the C-reactive proteins from different species, which are structurally highly conserved, the capacity for selective binding to apoB-containing plasma lipoproteins is also a constant feature. These interactions may therefore be related to the in vivo function of CRP in all species and this function may in turn be relevant to pathological conditions, such as atherosclerosis, in which lipoproteins are important.

Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.

Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.

Variation in lipoprotein(a) concentration associated with different apolipoprotein(a) alleles.

Plasma lipoprotein(a) (Lp(a)) concentrations vary considerably between individuals. To examine the variation for products of the same and different apolipoprotein(a) (apo(a)) alleles, conditions were established whereby phenotyping immunoblots could be used to estimate the concentration of Lp(a) associated with the constituent apo(a) isoforms. In these studies 28 distinct isoforms were identified, each differing by a single kringle IV unit. Tracking the isoforms through 10 families showed that there could be up to 200-fold difference in the Lp(a) concentration associated with the same-sized isoform produced from different alleles. In contrast there was typically < 2.5-fold variation in the Lp(a) concentration associated with the same allele. However, there were four occasions where the concentration associated with a particular allele was reduced below the typical range from one generation to the next. A nonlinear, inverse trend with isoform size was apparently superimposed upon the other factors that determine Lp(a) concentration. Inheritance of familial hypercholesterolemia or familial-defective apoB100 had little consistent effect upon Lp(a) concentration. In both the families and in other unrelated individuals the distribution of isoforms and their associated concentrations provided evidence for the presence of at least two and possibly more subpopulations of apo(a) alleles with different sizes and expression.

Relationship between apolipoprotein(a) phenotype, lipoprotein(a) concentration in plasma, and low density lipoprotein receptor function in a large kindred with familial hypercholesterolemia due to the pro664----leu mutation in the LDL receptor gene.

In a large kindred of 66 individuals, 22 were identified as heterozygous and 3 as homozygous for a mutation (pro664----leu) in the LDL-receptor gene that gives rise to familial hypercholesterolaemia (FH). All the heterozygotes had a raised level of plasma total cholesterol and low density lipoprotein cholesterol, but were remarkably free from premature coronary disease. Determination of apolipoprotein(a) (apo(a)) phenotype and lipoprotein(a) (Lp(a)) concentration in plasma revealed that in many instances, involving individuals with various apo(a) phenotypes, there was no difference in plasma Lp(a) concentration between an FH heterozygote and an unaffected sibling with the same apo(a) phenotype. No significant difference in Lp(a) concentration was observed between groups of FH and non-FH of the same apo(a) phenotype, although in each case the mean value for the FH group was greater than that for the non-FH group. There was also evidence for an inherited trait that markedly increased Lp(a) concentration, which did not segregate with apo(a) phenotype or the defective LDL-receptor allele. The data provide no evidence for a strong multiplicative interaction between the gene loci for apo(a) and the LDL receptor.

Metabolic basis of hyperapobetalipoproteinemia. Turnover of apolipoprotein B in low density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia.

The turnover of apolipoprotein B (apo B) in very low density, intermediate density, and low density lipoproteins (VLDL, IDL, and LDL) and in the light and heavy fractions of LDL was determined in seven patients with hyperapobetalipoproteinemia (hyperapo B), six normolipidemic subjects, and five patients with heterozygous familial hypercholesterolemia (FH). After receiving an injection of 125I-VLDL, hyperapo B patients were found to have a higher rate of synthesis of VLDL-apo B than controls (40.1 vs. 21.5 mg/kg per d, P less than 0.05) but a reduced fractional catabolic rate (FCR) (0.230 vs. 0.366/h, P less than 0.01). After receiving an injection of 131I-LDL, hyperapo B patients had higher rates of LDL-apo B synthesis than controls (23.1 vs. 13.0 mg/kg per d, P less than 0.001), as did FH patients (22.7 mg/kg per d). The FCR of LDL was similar in hyperapo B patients and controls (0.386 vs. 0.366/d) but was markedly decreased in FH patients (0.192/d). Most subjects exhibited precursor-product relationships between VLDL and IDL, and all did between IDL and light LDL; an analogous relationship between light and heavy LDL was evident in most hyperapo B patients and controls but not in FH patients. Simultaneous injection of differentially labeled LDL fractions and deconvolution analysis showed increased light LDL synthesis with normal conversion into heavy LDL in hyperapo B, whereas in FH conversion of light LDL was reduced and there was independent synthesis of heavy LDL. These data show that the increased concentration of LDL-apo B in hyperapo B is solely due to increased LDL synthesis, which is secondary to increased VLDL synthesis; in contrast, in FH there is both an increase in synthesis of LDL (which is partly VLDL-independent) and reduced catabolism.

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.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk.

AIMS: To determine the relative frequency of mutations in three different genes (low-density lipoprotein receptor (LDLR), APOB, PCSK9), and to examine their effect in development of coronary heart disease (CHD) in patients with clinically defined definite familial hypercholesterolaemia in UK. PATIENTS AND METHODS: 409 patients with familial hypercholesterolaemia patients (158 with CHD) were studied. The LDLR was partially screened by single-strand conformational polymorphism (SSCP) (exons 3, 4, 6-10 and 14) and by using a commercial kit for gross deletions or rearrangements. APOB (p.R3500Q) and PCSK9 (p.D374Y) were detected by specific assays. Coding exons of PCSK9 were screened by SSCP. RESULTS: Mutations were detected in 253 (61.9%) PATIENTS: 236 (57.7%) carried LDLR, 10 (2.4%) carried APOB p.Q3500 and 7 (1.7%) PCSK9 p.Y374. No additional mutations were identified in PCSK9. After adjusting for age, sex, smoking and systolic blood pressure, compared to those with no detectable mutation, the odds ratio of having CHD in those with an LDLR mutation was 1.84 (95% CI 1.10 to 3.06), for APOB 3.40 (0.71 to 16.36), and for PCSK9 19.96 (1.88 to 211.5; p = 0.001 overall). The high risk in patients carrying LDLR and PCSK9 p.Y374 was partly explained by their higher pretreatment cholesterol levels (LDLR, PCSK9 and no mutation, 10.29 (1.85), 13.12 and 9.85 (1.90) mmol/l, respectively, p = 0.001). The post-statin treatment lipid profile in PCSK9 p.Y374 carriers was worse than in patients with no identified mutation (LDL-C, 6.77 (1.82) mmol/l v 4.19 (1.26) mmol/l, p = 0.001, HDL-C 1.09 (0.27) mmol/l v 1.36 (0.36) mmol/l, p = 0.03). CONCLUSIONS: The higher CHD risk in patients carrying PCSK9 p.Y347 or a detected LDLR mutation supports the usefulness of DNA testing in the diagnosis and management of patients with familial hypercholesterolaemia. Mutations in PCSK9 appear uncommon in patients with familial hypercholesterolaemia in UK.

Abnormal structure and co-operative binding of low-density lipoprotein receptors containing the Glu-80-->Lys mutation.

The properties of low-density lipoprotein (LDL) receptors containing a Glu to Lys substitution at position 80 have been studied in fibroblasts from a homozygous familial hypercholesterolaemic subject (MB) and in monkey COS cells transfected with the mutant cDNA. Receptors containing the Glu-80-->Lys mutation were processed more slowly than the normal protein and only approx. 50% reached the surface as the mature form. Both cell types exhibited a normal concentration binding curve for beta-very-low-density lipoproteins (beta-VLDL) but an atypical, sigmoidal curve for LDL. The mature mutant receptor protein migrated abnormally slowly on SDS-PAGE under non-reducing conditions but normally under reducing conditions or after treatment with neuraminidase. It also showed an unusual ability to form dimers that were stable in detergents. Transfected normal and mutant receptors were apparently cleaved on the surface of the cells to give a product lacking the NH2-terminal portion of the protein, which was resistant to further proteolytic digestion. The results suggest that the Glu-80-->Lys substitution produces a change in the conformation of the protein, stabilized by polysaccharide chains, which results in a strong self-association of receptor molecules that affects their ability to bind LDL.

Determinants of variable response to statin treatment in patients with refractory familial hypercholesterolemia.

Interindividual variability in low density lipoprotein (LDL) cholesterol (LDL-C) response during treatment with statins is well documented but poorly understood. To investigate potential metabolic and genetic determinants of statin responsiveness, 19 patients with refractory heterozygous familial hypercholesterolemia were sequentially treated with placebo, atorvastatin (10 mg/d), bile acid sequestrant, and the 2 combined, each for 4 weeks. Levels of LDL-C, mevalonic acid (MVA), 7-alpha-OH-4-cholesten-3-one, and leukocyte LDL receptor and hydroxymethylglutaryl coenzyme A reductase mRNA were determined after each treatment period. Atorvastatin (10 mg/d) reduced LDL-C by an overall mean of 32.5%. Above-average responders (LDL-C -39.5%) had higher basal MVA levels (34.4+/-6.1 micromol/L) than did below-average responders (LDL-C -23.6%, P<0.02; basal MVA 26.3+/-6.1 micromol/L, P<0.01). Fewer good responders compared with the poor responders had an apolipoprotein E4 allele (3 of 11 versus 6 of 8, respectively; P<0.05). There were no baseline differences between them in 7-alpha-OH-4-cholesten-3-one, hydroxymethylglutaryl coenzyme A reductase mRNA, or LDL receptor mRNA, but the latter increased in the good responders on combination therapy (P<0.05). Severe mutations were not more common in poor than in good responders. We conclude that poor responders to statins have a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption, possibly mediated by apolipoprotein E4. If so, statin responsiveness could be enhanced by reducing dietary cholesterol intake or inhibiting absorption.

Non-saturable degradation of LDL by monocyte-derived macrophages leads to a reduction in HMG-CoA reductase activity with little synthesis of cholesteryl esters.

In monocyte-derived macrophages from both normal and familial hypercholesterolaemic (FH) subjects, degradation of low density lipoprotein (LDL) through non-saturable pathways produced the same fall in 3-hydroxy-3-methylglutaryl-CoA reductase activity as receptor-mediated degradation of acetylated LDL, yet did not lead to as great an increase in incorporation of [14C]oleate into cholesteryl esters. Studies using FH cells showed that the simultaneous addition of LDL did not reduce oleate incorporation resulting from degradation of acetylated LDL, and that there was a similar relationship for both lipoproteins between the increase in net oleate incorporation and the increase in the cholesteryl ester content of the cells. FH cells maintained in serum-free medium accumulated more free cholesterol than cells in lipoprotein-deficient serum when incubated with LDL but not when incubated with acetylated LDL. The results suggest that the cholesterol released from non-saturable degradation of LDL is more easily removed from the cells by acceptors in the medium than cholesterol released from receptor-mediated uptake of acetylated LDL, and is not readily available for esterification.

Binding and degradation of heavy and light subfractions of low density lipoprotein by cultured fibroblasts and macrophages.

The heavy and light subfractions of low density lipoprotein (LDL) were bound to the same extent and with the same affinity by the LDL receptors of cultured human fibroblasts, both when assayed at 4 degrees C and when assayed at 37 degrees C. They were also degraded similarly by the low affinity, LDL-receptor-mediated pathway exhibited by normal human monocyte-derived macrophages maintained in medium containing whole serum. Neither of the subfractions was taken up by the 'scavenger' pathway in mouse peritoneal or human monocyte-derived macrophages. Assuming that the LDL particles were not altered during isolation, the results provide no evidence to suggest that the higher fractional catabolic rate of light LDL observed in vivo can be explained by any preferential catabolism through LDL-receptor-mediated pathways.

Metabolism of apolipoprotein B-containing lipoproteins in familial hypercholesterolaemia: effects of plasma exchange.

The turnover of apolipoprotein B (apo B) in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) was investigated in 2 homozygous and 3 heterozygous patients with familial hypercholesterolaemia. The effects of a marked reduction in plasma LDL concentration, brought about by plasma exchange, upon apo B turnover were studied in 4 patients. Specific activity-time curves for the the plasma apo B after intravenous radioactive VLDL before plasma exchange indicated that in the heterozygotes all IDL-apo B was derived from VLDL and all LDL-apo B was derived from IDL, but the curves from the homozygotes showed that a significant fraction of the LDL in the plasma was not derived from IDL. Plasma exchange did not increase the rates of synthesis of LDL-apo B or VLDL-apo B and had no significant effect on the precursor--product relationship between IDL-apo B and LDL-apo B in heterozygous or homozygous patients. These findings provide no support for the hypothesis that apo B synthesis is controlled by the plasma LDL.

The metabolism of very low density and intermediate density lipoproteins in patients with familial hypercholesterolaemia.

The metabolism of apolipoprotein B (apoB) in very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) was studied in normal subjects and in patients with familial hypercholesterolaemia (FH) after an intravenous injection of autologous VLDL labelled with 125I. There were no significant differences in half life, pool size and turnover rate (mg/kg/h) of VLDL-apoB between the normal subjects, the FH heterozygotes and the FH homozygotes. IDL-apoB metabolism in the FH patients differed significantly from that in the normal subjects. In the FH patients, the rise to the maximum of the specific activity curve was slower, the half life of the descending limb of the specific-activity curve was longer, the fractional rate of turnover was lower and the plasma concentration was higher than in the normals. The effect of cholestyramine on IDL-apoB metabolism in the normal subjects did not differ from that in the FH heterozygotes and homozygotes, though cholestyramine is known to stimulate hepatic uptake of low density lipoprotein (LDL) by the LDL receptor. It is suggested that in normal human subjects the LDL receptor makes some contribution to the hepatic uptake of IDL-apoB derived from VLDL, but that IDL uptake is mediated partly by a separate receptor that recognizes apolipoprotein E but not apoB.

Catabolism of lipoprotein(a) in familial hypercholesterolaemic subjects.

The in vivo turnover of autologous lipoprotein(a) (Lp(a)) was studied in four heterozygous familial hypercholesterolaemic (FH) subjects and four subjects who were hyperlipidaemic but not FH. Each of the FH subjects exhibited a much lower fractional catabolic rate (FCR) for LDL than each of the non-FH subjects. Lp(a) was purified by sequential density gradient centrifugations and was radio-iodinated. The labelled Lp(a) ran as a single band on electrophoresis in gradient polyacrylamide gels. Less than 5% of the label was in lipid, with about 40% of the remainder on apolipoprotein B (apo B) and 60% on apo(a). Labelled and unlabelled Lp(a) competed equally poorly with LDL for binding to LDL receptors on cultured fibroblasts. The FCR of Lp(a), calculated from the decay of the specific radioactivity of the Lp(a) isolated from the daily blood samples, was the same in FH subjects as in non-FH subjects. There was no consistent relationship between Lp(a) FCR and the plasma Lp(a) concentration or between FCR and the Lp(a) phenotype, at least within this sample of subjects. There was a strong association between Lp(a) concentration and production rate, with values for non-FH and FH subjects falling on the same line. The rate of decline of radioactivity in whole plasma was consistently slower than the fall in specific radioactivity of the isolated Lp(a). This difference was more marked in FH subjects than in non-FH subjects and resulted from the accumulation of radioactivity derived from the injected Lp(a) at a lower density than Lp(a), in the fractions containing LDL. The amount of radioactivity in this fraction increased for the first few days after injection and then fell, the fall being more rapid in non-FH than in FH subjects. These results provide no evidence for the involvement of LDL receptors in the catabolism of Lp(a) itself but suggest that they could be responsible for some of the clearance of the lipid and apo B components after removal of apo(a) in the circulation.

Lipoprotein(a) in subjects with familial defective apolipoprotein B100.

The plasma lipoprotein(a) (Lp(a)) concentration and apolipoprotein(a) (apo(a)) phenotype were determined in the members of two families affected with familial defective apo B100 (FDB), resulting from the Arg3500----Gln mutation in apo B that disrupts binding to LDL receptors. Eleven different phenotypic species of apo A were identified, five of which were present in both families. Although there was a general increase in Lp(a) concentration as the size of the predominant apo(a) component decreased, there was considerable variability and in three clear instances the concentration of an inherited phenotypic species was atypically low. In five cases where a direct comparison could be made, the plasma Lp(a) concentration was significantly higher in heterozygous FDB subjects than in their non-FDB siblings or close relatives with the same phenotype. However, in vitro competition studies using purified Lp(a) that had been reduced with dithiothreitol to remove the apo(a) component, indicated that the Lp(a) from FDB heterozygotes contained a smaller proportion of defective particles than their LDL. Lp(a) particles containing normal and binding-defective apo B were present at approximately the same concentration, suggesting that the increase in Lp(a) concentration observed in FDB subjects could not be explained by the inability of the particles containing the defective apo B100 to be cleared through LDL-receptor mediated processes.

Influence of genotype at the low density lipoprotein (LDL) receptor gene locus on the clinical phenotype and response to lipid-lowering drug therapy in heterozygous familial hypercholesterolaemia. The Familial Hypercholesterolaemia Regression Study Group.

The relationship between molecular defect and clinical phenotype has been examined in 42 patients with heterozygous familial hypercholesterolaemia (FH) and premature coronary heart disease. The defined defects included mutations in the low density lipoprotein (LDL)-receptor gene (23/42) or the apolipoprotein B Arg3500Gln mutation (5/42). Mean LDL-cholesterol was higher, both before and during treatment with simvastatin and bile acid sequestrants, in patients predicted as having a 'severe' mutation than in those with a 'mild' mutation (8.72 +/- 2.02 mmol/l vs 6.63 +/- 1.8, P = 0.05 before and 4.51 +/- 0.90 mmol/l vs 3.19 +/- 0.58, P = 0.05 during treatment). Maximum inducible LDL-receptor activity in cultured lymphoblasts was inversely correlated with LDL-cholesterol before (r2 = 0.499, P = 0.002) and during (r2 = 0.478, P = 0.004) treatment in patients with a defined mutation in the LDL-receptor gene, but not in the 14 patients with no detectable molecular defect. LDL-cholesterol concentrations before and during treatment were significantly correlated in patients with a defined LDL-receptor gene mutation (r2 = 0.548, P = 0.0001), but not in those with no detectable genetic defect. All these correlations were weak, however and there were no differences in the response to treatment in terms of either relative reduction or absolute decrease in LDL-cholesterol concentration between patients with different LDL-receptor defects. We conclude that only part of the variable phenotype of heterozygous FH patients is explained by different LDL-receptor defects and that other factors determine the severity of their hypercholesterolaemia and the onset of coronary disease.

Detection and quantitation of apolipoprotein(a) mRNA in human liver and its relationship with plasma lipoprotein(a) concentration.

Northern blotting and hybridisation with specific probes was used to detect and quantitate apolipoprotein(a) (apo(a)) mRNA in total RNA isolated from 25 human liver samples. A total of 14 different transcripts were identified suggesting that there are at least 15 different alleles at the apo(a) locus including a probable null allele. Apo(a) mRNA sizes were linearly correlated with the electrophoretic mobility of plasma apo(a) glycoprotein isoforms, and differed, in many cases, by the equivalent of one Kringle 4 unit. To investigate the relationship between apo(a) mRNA size and its concentration in the liver, and between hepatic apo(a) mRNA concentration and plasma lipoprotein(a) (Lp(a)) levels, apo(a) mRNA was quantified by densitometric scanning of autoradiograms of Northern blots. Overall, there was a significant inverse correlation between apo(a) mRNA size and its concentration in the liver, despite a marked interindividual variability in the relative amounts of similar-sized transcripts. In each heterozygous individual, the difference in concentration between the two mRNA species was determined by the difference in size. However, there was not a significant relationship between hepatic apo(a) mRNA concentration and plasma Lp(a) levels in this group. These findings emphasise the importance of mechanisms other than the rate of transcription of the apo(a) gene in the regulation of Lp(a) synthesis.

Cholesterol-lowering drug therapy in a patient with receptor-negative homozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is caused by mutations in the gene for the low density lipoprotein (LDL) receptor. It is generally believed that homozygous FH patients do not respond well to lipid-lowering drug therapy with inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase because they cannot respond to an increased demand for hepatic cholesterol by up-regulation of LDL-receptor activity. In this paper we show that serum cholesterol in a homozygous FH patient with a receptor-negative LDL-receptor phenotype was reduced by 30% after treatment with simvastatin alone and by a further 11% with simvastatin in combination with probucol and nicotinic acid. The patient was a true homozygote, with two identical alleles of the LDL receptor gene in which a previously undescribed point mutation in exon 11 introduces a premature termination codon at residue 540 in the protein; the mutant protein is predicted to be truncated in the domain with homology to the epidermal growth factor precursor. Cultured cells from the patient were unable to bind, internalise or degrade LDL by the receptor pathway and there was no immunodetectable LDL receptor protein in the cells. Thus the lipid lowering effect of simvastatin in this individual must involve mechanisms other than stimulation of LDL receptors.