Estimation of the age of the ancestral arginine3500-->glutamine mutation in human apoB-100.

Familial defective apoB-100 (R3500Q) [FDB (R3500Q)] is caused by a mutation in the apoB gene (2p23.24). Almost all individuals with this disorder are of European descent, and in almost all cases the mutation is on a chromosome with a rare haplotype (194) at the apoB locus, suggesting that all FDB (R3500Q) probands are descended from a common ancestor in whom the original mutation occurred. The distribution of the mutation is consistent with an origin in Europe 6000-7000 years ago. We have estimated the amount of recombination between the apoB gene and markers on chromosome 2 in 34 FDB (R3500Q) probands in whom the mutation is on a 194 haplotype. Significant linkage disequilibrium was found between the apoB gene and marker D2S220. We have identified three YACs that contain the apoB gene and D2S220. The shortest restriction fragment common to the three YACs that contained both loci was 240 kb long. No shorter fragments with both loci were identified. On the assumption that 1000 kb corresponds to 1 cM, we deduce that the recombination distance between D2S220 and the apoB gene is about 0.24 cM. Combining this value with the linkage disequilibrium observed between the two loci in the probands, we estimate that the ancestral mutation occurred about 270 generations ago. We postulate that the original mutation occurred in the common ancestor of living FDB (R3500Q) probands, who lived in Europe about 6750 years ago. The errors in this estimate are discussed.

Does the presence of the 3500 mutant apolipoprotein B-100 in low density lipoprotein particles affect their atherogenicity?

Apolipoprotein B-100 (apo B-100) is the protein component of low density lipoprotein (LDL) responsible for its binding and clearance by LDL receptors (LDL-R). In familial defective apo B-100 (FDB), a mutation in apo B-100 at residue 3500 markedly reduces its affinity for LDL-R, often causing accumulation of defective LDL particles, and an increased proneness to coronary artery disease (CAD). In FDB heterozygotes, about 70% of the LDL particles are mutant, which may alter their atherogenicity relative to LDL containing normal apo B. Therefore, we compared CAD in heterozygous FDB with CAD in heterozygous familial hypercholesterolemia (FH), since raised LDL is usually present from birth in both conditions, and in FH the LDL particles that accumulate have normal apo B, as the inherited defect involves the LDL-R. The clinical presentation of coronary atherosclerosis and its angiographic appearance were examined in FDB and FH patients matched for conventional cardiac risk factors (hypertension, smoking, sex) and serum lipid levels. There was no significant difference between the FDB and FH patients (n=11 pairs) in the type of cardiac symptoms or their ages of onset (50 +/- 9 vs. 45 +/- 11 years). Coronary angiographic appearance was also similar in both groups (n=9 pairs). These observations suggest that LDL particles with the 3500 mutation in apo B have the same atherogenicity as LDL particles with normal apo B.

The affinity of low-density lipoproteins and of very-low-density lipoprotein remnants for the low-density lipoprotein receptor in homozygous familial defective apolipoprotein B-100.

We have identified two familial defective apo B-100 (FDB) homozygotes by DNA sequencing and have measured affinity of low-density lipoproteins (LDL) and very-low-density lipoprotein (VLDL) remnants for the LDL receptor in vitro. The patients were a 66-year-old man with coronary heart disease (plasma cholesterol level, 9.5 mmol/l before treatment) and his 69-year-old sister, without signs of cardiovascular disease (plasma cholesterol, 12.0 mmol/l before treatment). In both patients, treatment with statins caused a marked fall in plasma cholesterol level. Binding affinity of LDL from the two patients was 10%-20% of normal at 4 degrees C and 37 degrees C. Binding affinity of VLDL remnants was normal. We conclude that (1) residual affinity of LDL in homozygous FDB is high enough to permit significant catabolism via the LDL-receptor pathway, and (2) normal affinity of VLDL remnants permits normal hepatic clearance of precursors of LDL and increased clearance of LDL precursors when receptor activity is stimulated by statins. Residual affinity of LDL and normal affinity of remnants could explain why expression of the FDB mutation is generally milder than that of LDL receptor mutations causing familial hypercholesterolaemia.

Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolaemia.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder caused by the substitution of glutamine for arginine at position 3500 in apo B-100. The presence of mutant apo B-100 in low-density lipoproteins (LDL) markedly reduces their affinity for the LDL receptor, leading to hypercholesterolaemia and increased proneness to coronary artery disease. In some FDB heterozygotes the clinical picture is indistinguishable from that in heterozygous familial hypercholesterolaemia (FH). In European and N. American populations the frequency of FDB is at least as high as that of FH. In most lipid clinics, 2-5% of patients given a clinical diagnosis of FH have FDB, not FH. Most FDB heterozygotes respond well to drugs that lower plasma LDL levels by inducing receptor activity. This may be due partly to increased receptor-mediated hepatic removal of mutant and normal precursors of LDL, using apo E as recognition element. Several important lessons can be learnt from the study of FDB.

The binding of very low density lipoprotein remnants to the low density lipoprotein receptor in familial defective apolipoprotein B-100.

We have compared the affinity for low density lipoprotein (LDL) receptors of LDL and very low density lipoprotein (VLDL) remnants from patients with familial defective apo B-100 (FDB) with that of LDL and VLDL remnants from normal subjects. The binding affinity of FDB LDL was markedly reduced in all 14 FDB patients examined, hut the affinity of FDB remnants did not differ significantly from that of remnants prepared from normal subjects. Since the mutant form of apo B-100 present in FDB is recognized by LDL receptors with greatly reduced efficiency, we suggest that apo B plays only a minor role in the receptor-mediated uptake of VLDL remnants by the liver in man. These results are consistent with our previous suggestion that the ability of drugs that stimulate hepatic receptor activity to lower the plasma LDL level in FDB is due in part to increased hepatic uptake of lipoprotein precursors of LDL, including remnant particles with normal apo B-100 and those with mutant apo B-100.

Variable expression of the mutation in familial defective apolipoprotein B-100.

Although most subjects with familial defective apolipoprotein B-100 (FDB) have raised plasma low-density lipoprotein (LDL) levels, a few have LDL levels within the normal range. We have previously identified two normocholesterolemic FDB heterozygotes in an affected family. Results obtained from a study of this family are compatible with a major genetic contribution to the normocholesterolemia in the two heterozygotes. However, our findings are not compatible with inheritance of a variant normal allele at the apolipoprotein B locus in this family that neutralizes the effect of an FDB allele on the plasma LDL level. Polymorphic variations at the apolipoprotein E and LDL receptor loci did not explain the presence of normal LDL levels in the two heterozygous FDB subjects.

Effective reduction of plasma LDL levels by LDL apheresis in familial defective apolipoprotein B-100.

The clinical response to long-term reduction of the plasma LDL cholesterol concentration was studied in a man with severe coronary artery disease associated with familial defective apolipoprotein B-100 (FDB). Plasma exchange repeated at 2-week intervals, combined with lipid-lowering drugs, led to remission of angina and improved exercise test performance. A similar clinical response was achieved after LDL apheresis with dextran sulphate columns repeated once every 2 weeks in combination with drug treatment. The reduction in plasma LDL cholesterol level brought about by LDL apheresis was at least as marked in the FDB patient as in 5 patients with familial hypercholesterolaemia. We conclude that FDB patients with coronary artery disease may derive clinical benefit from prolonged reduction of their plasma cholesterol levels and that LDL containing apo B-100 in which arginine at position 3500 is replaced by glutamine is removed from plasma by dextran sulphate columns as efficiently as is normal LDL.

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.

Does the EcoRI polymorphism in the human apolipoprotein B gene affect the binding of low density lipoprotein to the low density lipoprotein receptor?

In human populations, there is an association between coronary artery disease and a polymorphism in the apolipoprotein B (apo B) gene detected with the enzyme EcoRI. This polymorphism gives rise to two apo B alleles, one (E+) encoding glutamic acid and the other (E-) encoding lysine at position 4,154 in apo B-100, the protein of low density lipoprotein (LDL). We have tested the hypothesis that this amino acid substitution indirectly influences proneness to coronary artery disease by affecting the binding of LDL to LDL receptors. The receptor-binding affinities of LDLs from eight pairs of subjects with genotypes E+/+ and E-/- who were matched for other apo B genotypes were determined in vitro. There was no significant difference between the binding affinities of LDLs from the two groups of subjects. Our results strongly suggest that the amino acid at position 4,154 does not influence the function of the receptor-binding domain in apo B-100 and that the association between coronary artery disease and the EcoRI polymorphism is not mediated by an effect of the polymorphism on serum LDL concentration. In view of our findings, it would be of interest to examine the effect of the amino acid substitution on the binding of LDL to arterial proteoglycans and on the oxidizability of LDL by cells in culture.

Response to cholesterol-lowering drugs in familial defective apolipoprotein B-100.

The effect of cholestyramine and simvastatin, given separately or in combination, on serum lipid concentrations in 11 patients with heterozygous familial defective apolipoprotein B-100 was compared with that in 11 matched patients with heterozygous familial hypercholesterolaemia. In both groups of patients there was a substantial fall in serum lipid levels in response to treatment. There were no significant differences between the reductions in serum total or low-density lipoprotein cholesterol levels in the two groups.

Clinical signs of familial hypercholesterolemia in patients with familial defective apolipoprotein B-100 and normal low density lipoprotein receptor function.

In a previous study (Tybjaerg-Hansen et al, Atherosclerosis 1990;80:235-242), we identified nine patients heterozygous for the apolipoprotein B (apo B) arginine-to-glutamine (Arg3,500----Gln) mutation (familial defective apolipoprotein B-100 [FDB]). Six of these had been diagnosed clinically as familial hypercholesterolemic (FH) heterozygotes. We have since examined low density lipoprotein (LDL) receptor function in the FDB index patients and in three of their families. Skin fibroblasts from seven of seven unrelated FDB patients from whom cell lines were established exhibited normal high-affinity binding and degradation of normal LDL in vitro. In the three families, a raised plasma LDL concentration did not segregate with a haplotype of two polymorphic restriction sites at the LDL receptor locus. We conclude that the clinical and biochemical signs of classical FH can occur in the presence of the FDB mutation and a normal LDL receptor gene. In a four-generation family with 11 proven or presumed FDB heterozygotes, expression of the mutation ranged from normal plasma LDL concentrations and no clinical signs in two individuals, to hypercholesterolemia and death from myocardial infarction at age 31. Variable expression of the FDB mutation could not be explained conclusively by variation in diet, body mass index, smoking habit, apo E genotype, or plasma Lp(a) concentration.

Current approaches to the genetics of coronary heart disease (CHD) including an account of work done at Hammersmith Hospital.

Genetic variability makes a significant contribution to CHD in Western populations. The major genetic component in CHD in the general population is polygenic; only a small fraction of the total genetic contribution is due to rare monogenic disorders such as FH. Two approaches to the investigation of the genetics of CHD are described. In the first, the frequencies of the alleles at polymorphic sites in the apoB gene were compared in unrelated normal and CHD men. There was a positive association between CHD and two of the alleles investigated (E- and X-). The significance of these findings is discussed. In the second approach, patients with a clinical diagnosis of FH were screened for the presence of a rare mutation in the apoB gene giving rise to defective LDL particles. The mutation was detected in 3-4% of the screened population. This suggest that the apoB mutation may produce a clinical syndrome indistinguishable from FH.

Familial defective apolipoprotein B-100: detection in the United Kingdom and Scandinavia, and clinical characteristics of ten cases.

Familial defective apolipoprotein B-100 (FDB) is a recently identified, dominantly inherited genetic disorder, which leads to increased serum concentration of low density lipoprotein (LDL) cholesterol with reduced affinity for the LDL receptor. This disorder is associated with a G to A mutation in exon 26 of the apolipoprotein B (apo B) gene which creates a substitution of glutamine for arginine in the codon for amino acid 3500. We have searched for this mutation in 374 unrelated individuals with hyperlipidaemia from the United Kingdom, and in 371 unrelated individuals with a primary clinical diagnosis of atherosclerosis from the United Kingdom and Scandinavia. Ten individuals, 9 from the U.K. and 1 from Denmark, were identified. The frequency of the mutation was 3% in individuals classified clinically as having familial hypercholesterolaemia (FH) and 3% in individuals with type IIa hyperlipidaemia without FH, and was not found in patients with types IIb and III hyperlipidaemia. The mutation was rare in individuals with a primary clinical diagnosis of atherosclerosis. Plasma lipid levels and clinical characteristics of the ten patients identified in the present study are similar to those reported for heterozygous FH. Thus, in our study, FDB is associated with moderate to severe hypercholesterolaemia, and appears to be a serious disorder causing premature cardiovascular disease. Individuals with this mutation can be identified unambiguously using routine molecular screening techniques.

Apolipoprotein A-I gene polymorphisms: frequency in patients with coronary artery disease and healthy controls and association with serum apo A-I and HDL-cholesterol concentration.

We have investigated the association between serum high density lipoprotein-cholesterol (HDL-C) and apo A-I concentration and the PstI and XmnI restriction fragment length polymorphisms of the apolipoprotein AI-CIII-AIV multigene complex. Two groups of subjects were examined. The first comprised 174 unrelated male patients under 60 years of age with angiographic evidence of coronary artery disease (CAD). Of this group 34 were non-North European. The second group consisted of 104 unrelated healthy male North European subjects aged under 60 and free from demonstrable CAD, who attended a health screening clinic in London. For the PstI polymorphism, the frequency of the rarer P2 allele was 0.12 in both the North European and non-North European patients and this was higher than in the control group (P2 frequency 0.06, P less than 0.05). Healthy individuals with the genotype P1P2 had higher levels of apo A-I but similar levels of HDL-C compared to those with the genotype P1P1. However, CAD patients with the genotype P1P2 had lower serum levels of apo A-I and significantly lower serum levels of HDL-C compared to those with the genotype P1P1 (0.85 mmol/l vs. 1.0 mmol/l, P less than 0.05). The allele frequencies of the XmnI polymorphisms were not significantly different in the control group and the group of North European patients, although within the sample of non-North European patients, the frequency of the X2 allele was significantly higher than that found in the North European controls (0.26 vs. 0.09). Patients with the genotype X1X2 had a higher mean serum concentration of HDL-C and apo A-I compared with patients with the genotype X1X1 (1.14 and 0.93 mmol/l for HDL-C, P less than 0.05; 147 and 123 mg/dl for apo A-I, P less than 0.05). Associations between HDL-C and apo A-I levels and PstI and XmnI genotype were similar in patients taking and not taking beta-blockers. The data show that genetic variation in the apo AI-CIII-AIV gene cluster is associated with coronary artery disease although only weakly, and suggest that the mechanism of this association may operate through an effect in determining the serum concentration of apo A-I and HDL-cholesterol.

Restriction fragment length polymorphisms in the apo B gene in relation to coronary artery disease.

We have determined the frequencies of the alleles at the EcoRI (E), XbaI (X) and PvuII (P) polymorphic restriction sites in the apo B gene in 124 white men with coronary artery disease (CAD) and in 146 white men free from CAD. The frequencies of the E- (restriction site absent) and X- alleles were both significantly higher in normocholesterolaemic men with CAD than in those without CAD, but the frequency of the P+ allele (restriction site present) was similar in the 2 groups. The frequency of the E- allele was significantly higher in CAD men with hypertriglyceridaemia than in normal men without hypertriglyceridaemia. In the normocholesterolaemic men without CAD, the mean serum cholesterol concentration was higher in those with genotype X++ than in those with genotype X--. Mean serum LDL-apo B and LDL-cholesterol concentrations did not differ significantly between men with different XbaI or EcoRI genotypes. Serum apo A-I levels differed significantly between normal men with different XbaI genotypes. Serum HDL-cholesterol levels differed significantly between CAD men with different XbaI genotypes. These results suggest that in white men the E- and X- alleles are in linkage disequilibrium with a nearby allele that is causally related to CAD. It is also possible that the amino acid substitution at position 4154 in apo B, brought about by the nucleotide change responsible for the EcoRI polymorphism, has a direct effect on the atherogenicity of LDL.

Molecular cloning of human LDL apolipoprotein B cDNA. Evidence for more than one gene per haploid genome.

We have isolated an apolipoprotein B (apo B) clone (pXB1) from a human liver cDNA expression library, by immunoselection with a polyclonal antibody to human low density lipoprotein. pXB1 was used to isolate 3 clones (pB2, pB3 and pB4), containing cDNA inserts spanning a region of 3.75 kbp, from a second human liver cDNA library. We report the sequence of 1359 nucleotides at the 3' end of the pB4 cDNA insert and the amino acids encoded by this sequence. The cDNA inserts of pBX1 and pB2 overlapped the sequenced portion of pB4. pB2 contained an EcoR1 restriction site (resulting in a Glu-Lys replacement) which is not present in pB3 or pB4 and pB3 contained an MspI site not present in pB2 or pB4. Since all 3 clones were derived from the mRNA of a single human liver, we suggest that the human haploid genome contains more than one functional apo B gene. Labelled probes spanning almost the whole of the pB4 cDNA insert hybridized with RNA from human liver and small intestine, showing that the apo B mRNAs from these two tissues have nucleotide sequences in common. The nucleotide sequence in human liver apo B mRNA is probably longer than 12 kb, showing that the MW of monomeric apo B is at least 350kd. Clone pB4 hybridized with mRNA of similar length in rabbit liver and small intestine. These results raise the possibility that the low MW apo B synthesized in the intestine (B-48) and the high MW apo B synthesized in the liver (B-100) are translated from the same mRNA. The expression products of fragments of pB4 cloned into an expression vector were blotted with monoclonal antibodies to human LDL. The results suggest that the cDNA insert in pB4 encodes a part of apo B common to B-48 and B-100 and a region close to the recognition site for the LDL receptor.

The distribution of cholesterol and apoprotein A-I between the lipoproteins in plasma and peripheral lymph from normal human subjects.

The lipoproteins of peripheral lymph and plasma from normal human subjects were separated according to their density by sequential ultracentrifugation and according to their size by gradient gel electrophoresis and gel exclusion chromatography. High density lipoproteins (HDL) carried a higher proportion of the total cholesterol in lymph than in plasma. Within the HDL fraction, the less dense and more lipid-rich component (HDL2) carried a higher proportion of the total HDL cholesterol in lymph than in plasma. Gradient gel electrophoresis showed (1) a higher proportion of large to small HDL particles in lymph than in plasma and (2) the presence of at least three populations of apo A-I-containing lipoproteins with Stokes diameters larger than the Stokes diameter of HDL2. Separation by gel exclusion chromatography showed that the proportion of large HDL particles with a high cholester: apo A-I ratio was greater in lymph than in plasma. In view of the sieving effect of the blood capillaries, which favours the passage across the capillary walls of smaller vs larger particles, we suggest that the higher ratio of large to small HDL particles in lymph than in plasma is due to the conversion of small to large HDL in the interstitial fluid by incorporation of cholesterol and other lipids from extravascular cells into the smaller particles.

Effects of starvation and plasma exchange on lecithin: cholesterol acyltransferase activity and cholesterol efflux in cholesterol-fed pigs.

The effects of starvation and of plasma exchange with a cholesterol-free substitute on efflux of tissue cholesterol and on lecithin: cholesterol acyltransferase (LCAT) activity in plasma and peripheral lymph were investigated in two pigs fed a cholesterol diet for 3-4 months. The pigs were labelled with i.v. [14C]cholesterol before plasma exchange or starvation. The cholesterol diet increased plasma total cholesterol concentration and LCAT activity in plasma and lymph, but had little effect on the rate of esterification of cholesterol in plasma or lymph. During cholesterol feeding, and when the animals were fed a normal diet, cholesterol esterification rates in plasma and lymph were much lower than the maximum rates achieved when LCAT was saturated with substrate, suggesting that LCAT in normal pig plasma and lymph is not saturated with substrate. Plasma exchange, carried out when the specific activity of tissue cholesterol exceeded that of plasma cholesterol, was followed by a brief rise in the specific activity of plasma cholesterol to a maximum value between the specific activities of muscle and adipose-tissue cholesterol, reflecting the transfer of radioactive cholesterol from tissue to plasma. During the rise in plasma total cholesterol specific activity there were no differences between the specific activities of low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in plasma or lymph. Starvation had no effect on the plasma-cholesterol specific-activity curve. From about day 14 after labelling, cholesterol-specific activity decreased in the order: tissues greater than lymph greater than plasma. This suggests that the transfer of cholesterol from tissues to plasma was mediated by lipoproteins in the interstitial fluid.