Rare variant of apolipoprotein E (Arg136-->Cys) in a subject with normal lipid values

During the screening of apolipoprotein (apo) E gene polymorphism with PCR and subsequent restriction analysis, we have identified a female carrier with a mutant allele Arg136-->Cys. This proband had normal lipid parameters and no history of coronary artery disease (CAD). We did not confirm the previously described connection between apo E Arg136-->Cys mutation and elevated lipid levels. In the case of this mutation, other factors (environmental and/or genetic) are important for the development of lipid metabolism disorders.     

Rare variant of apolipoprotein E (Arg136 -->Ser) in two normolipidemic individuals

Through the analysis of the common apolipoprotein (apo) E gene polymorphism in large Caucasian population study with the PCR and subsequent restriction analysis, we have identified carriers of mutant allele Arg136-->Ser. Both of them (71-years-old female and her 43-years-old son) have normal lipid parameters. We suggest that Arg136-->Ser mutation in apoE is not necessarily connected with elevated lipid levels in all cases. Furthermore, so far unidentified factors (environmental and/or genetic) are important for the development of lipid metabolism disorders in apoE Arg136-->Ser mutation carriers.     

The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia.

Type III hyperlipoproteinemia typically is associated with homozygosity for apolipoprotein (apo) E2(Arg158----Cys). Dominant expression of type III hyperlipoproteinemia associated with apoE phenotype E3/3 is caused by heterozygosity for a human apoE variant, apoE3(Cys112----Arg, Arg142----Cys). However, this apoE3 variant was not separable from the normal apoE3 in these patients' plasma because the two proteins have identical amino acid composition, charge, and molecular weight. Therefore, to determine the functional characteristics of this protein, we used recombinant DNA techniques to produce this apoE variant in bacteria. We also produced a non-naturally occurring variant, apoE(Arg142----Cys), that had only the cysteine substituted at residue 142. These two apoE variants were purified from cell lysates of the transfected Escherichia coli by ultracentrifugal flotation in the presence of phospholipid, by gel filtration chromatography, and by heparin-Sepharose chromatography. Both Cys142 apoE variants bound to lipoprotein receptors on human fibroblasts with only about 20% of normal binding activity. Therefore, cysteine at residue 142, not arginine at residue 112, is responsible for the decreased receptor binding activity of the variants. Cysteamine treatment and removal of the carboxyl-terminal domain had little effect on the binding activity, whereas both modulate the receptor binding activity of apoE2(Arg158----Cys). The mutation at residue 142 decreased the binding activity of apoE to both heparin and the monoclonal antibody 1D7 (this antibody inhibits receptor binding of apoE), whereas apoE2(Arg158----Cys), which is associated with recessive expression of type III hyperlipoproteinemia, binds normally to both. The Arg112, Cys142 variant predominantes 3:1 over normal apoE3 in the very low density lipoproteins of plasma from an affected subject, as assessed by differential reactivity with the antibody 1D7. The unique combination of functional properties of the Arg112, Cys142 variant provides a possible explanation for its association with dominant expression of type III hyperlipoproteinemia.     

Unmasking of Type III Hyperlipoproteinemia by Hypothyroidism: A Dramatic Illustration of Altered Lipoprotein Metabolism in a Postpartum Woman

ObjectiveTo illustrate the potential abnormalities in lipoprotein metabolism associated with type III hyperlipoproteinemia and the modulation of their clinical expression by thyroid hormone and estrogenic status.MethodsAn illustrative case, with associated clinical and laboratory data, is presented, and relevant clinical and pathophysiologic studies from the literature are reviewed.ResultsA 35-year-old woman, at 7 months after delivery of her first child, presented to her family physician with a complaint of painful eruptions on the palms of her hands. On evaluation, she was found to have new hypothyroidism and severe hypertriglyceridemia (> 1,569 mg/dL). Thyroxine replacement was initiated, and she was referred to the lipid clinic. When seen in the lipid clinic shortly thereafter, her triglyceride level had normalized, but her low-density lipoprotein (LDL) fraction was strikingly elevated (representing a combination of elevated intermediate-density lipoprotein and LDL cholesterol). On physical examination, palmar xanthomas were noted, suggestive of type III hyperlipoproteinemia. This diagnosis was further supported by homozygosity at the apolipoprotein E (apo E) gene locus for the apo E2 allele implicated in this condition. Ultimately, with attainment of euthyroidism in the subsequent weeks, the lipid profile normalized, with the LDL cholesterol concentration particularly reduced at 55 mg/dL.Conclusion: Clinical expression of type III hyperlipoproteinemia necessitates interaction between an underlying genetic defect of lipoprotein metabolism (apo E2 homozygosity) and a secondary metabolic insult such as, in the current case, hypothyroidism and possibly breast-feeding-mediated hypoestrogenemia. As such, in patients with type III hyperlipoproteinemia, it is essential to search for exacerbating factors, particularly because the amelioration of such factors may rectify the effects of the underlying dyslipidemia. (Endocr Pract. 2005;11:394-398)     

Characterization of five new mutants in the carboxyl-terminal domain of human apolipoprotein E: No cosegregation with severe hyperlipidemia

Assessment of the apolipoprotein E (apoE) phenotype by isoelectric focusing of both hyperlipidemic and normolipidemic individuals identified five new variants. All mutations were confined to the downstream part of the APOE gene by using denaturing gradient gel electrophoresis (DGGE). Sequence analysis revealed five new mutations causing unique amino acid substitutions in the carboxyl-terminal part of the protein containing the putative lipid-binding domain. Three hyperlipoproteinemic probands were carriers of the APOE*2(Val236→Glu) allele, the APOE*3(Cys112→Arg; Arg251→Gly) allele, or the APOE*1(Arg158→Cys; Leu252→Glu) allele. DGGE of the region encoding the receptor-binding domain was useful for haplotyping the mutations at codons 112 and 158. Family studies failed to demonstrate cosegregation between the new mutations and severe hyperlipoproteinemia, although a number of carriers for the APOE*3(Cys112→Arg; Arg251→Gly) allele and the APOE*1(Arg158→Cys; Leu252→Glu) allele expressed hypertriglyceridemia and/or hypercholesterolemia. Two other mutant alleles, APOE*4⁻ (Cys112→Arg; Arg274→His) and APOE*4⁺(Ser296→Arg), were found in normolipidemic probands. The lack of cosegregation of these new mutations with severe hyperlipoproteinemia suggests that these mutations do not exert a dominant effect on the functioning of apoE.     

Familial dysbetalipoproteinemia in three patients with apoE 2*(Arg136-->Cys) gene variant

Apolipoprotein E (apoE) is a polymorphic protein which occurs in three common isoforms and more than 25 rare variants. Some of the rare apoE variants have been implicated in a dominant mode of inheritance of familial dysbetalipoproteinemia (FD). We have identified three unrelated apoE 2*(Arg136-->Cys) carriers with FD. This finding supports the notion that although apoE 2*(Arg136-->Cys) mutation is perhaps not sufficient to cause FD itself, the presence of other genetic and/or environmental factors can lead to the phenotypic expression of the disease in the carriers.     

Apolipoprotein E3-Leiden. A new variant of human apolipoprotein E associated with familial type III hyperlipoproteinemia

Summary A variant of apolipoprotein E, denoted apo E3-Leiden, has been identified in a 41-year-old male suffering from type III hyperlipoproteinemia with xanthomatosis. Apo E3-Leiden focus in the E3 position. In contrast with normal apo E3, apo E3-Leiden is defective in binding to the low density lipoprotein (LDL) receptor and does not contain cysteine as evaluated by cysteamine treatment of very low density lipoprotein followed by isoelectric focusing and conventional protein staining and by amino acid analysis. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, apo E3-Leiden displays an electrophoretic mobility intermediate to that of normal apo E3 and apo E2 (Arg₁₅₈Cys). The mother and four siblings of the proband also have apo E3-Leiden and hyperlipoproteinemia type III; three of them with xanthomatosis. Two siblings do not show apo E3-Leiden in their VLDL fraction and do not have hyperlipoproteinemia type III. In the VLDL fractions of all affected family members only the presence of apo E3-Leiden could be detected after cysteamine treatment and isoelectric focusing followed by conventional protein staining. However, isoelectric focusing of cysteaminetreated sera followed by immunoblotting, using anti-apo E antiserum as first antiserum, demonstrates the presence of low amounts of normal apo E3 in addition to apo E3-Leiden in serum of the affected family members. These results indicate that all affected family members are heterozygotes E3/E3-Leiden and suggest that in this family type III hyperlipoproteinemia is transmitted as a dominant trait.     

A case of hyperlipidemia with homozygous apolipoprotein E5 (Glu3-->Lys)

In this study, we present clinical feature of a novel case with homozygous apolipoprotein (apo) E5.The patient was a 53-year-old Japanese woman. She was from a small island off the coast of Kagoshima Prefecture, Japan. Her parents were first degree cousins. No corneal opacification, xanthomatosis, lymphadenopathy, or hepatosplenomegaly was observed. There have been no signs of clinically overt atherosclerosis to date. Her serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL)-cholesterol levels were 11.6, 6.1 and 1.2 mmol/l, respectively, and apo A-I, A-II, B, C-II, C-III and E levels were 121, 34.8, 269, 10.4, 25.7 and 10.3 mg/dl, respectively. Serum lipoprotein profile analyzed by agarose gel electrophoresis and differential staining revealed markedly increased cholesterol and TG in both beta and prebeta-migrated lipoproteins, whereas alpha-migrated lipoprotein showed decreased cholesterol. Her apo E isoform analyzed by isoelectric focusing (IEF) was found to be homozygous apo E5.Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of her apo E and lipoprotein lipase (LPL) genes revealed that she had a homozygous apo E (Glu3-->Lys) and heterozygous LPL variant Ser447 to Ter. Her son and daughter, both of whom had hyperlipidemia, were found to have apo E3/5 phenotype. Direct sequencing analysis of her apo E gene confirmed a homozygous one nucleotide change: G to A at nucleotide position of 2836 in the exon 3, resulting in Glu3-->Lys mutation.This is the first report of lipids and lipoprotein profiles in patients with homozygous apo E5 (Glu3-->Lys).     

Molecular basis of familial chylomicronemia: mutations in the lipoprotein lipase and apolipoprotein C-II genes.

The molecular basis of familial chylomicronemia (type I hyperlipoproteinemia), a rare autosomal recessive trait, was investigated in six unrelated individuals (five of Spanish descent and one of Northern European extraction). DNA amplification by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis allowed rapid identification of the underlying mutations. Six different mutant alleles (three of which are previously undescribed) of the gene encoding lipoprotein lipase (LPL) were discovered in the five LPL-deficient patients. These included an 11 bp deletion in exon 2, and five missense mutations: Trp 86 Arg (exon 3), His 136 Arg (exon 4), Gly 188 Glu (exon 5), Ile 194 Thr (exon 5), and Ile 205 Ser (exon 5). The Trp 86 Arg mutation is the only known missense mutation in exon 3. The other missense mutations lie in the highly conserved "central homology region" in close proximity with the catalytic site of LPL. These and other previously reported missense mutations provide insight into structure/function relationships in the lipase family. The missense mutations point to the important role of particular highly conserved helices and beta-strands in proper folding of the LPL molecule, and of certain connecting loops in the catalytic process. A nonsense mutation (Arg 19 Term) in the gene encoding apolipoprotein C-II (apoC-II), the cofactor of LPL, was found to underlie chylomicronemia in the sixth patient who had normal LPL but was apoC-II-deficient.     

Allele-specific and asymmetric polymerase chain reaction amplification in combination: a one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100.

We have combined the asymmetric polymerase chain reaction (PCR) with allele-specific PCR to detect a single point mutation. A set of two priming oligonucleotides and a third allele-specific primer were used to identify heterozygotes for a G to A mutation at nucleotide 10,708 in the apolipoprotein B (apo B) gene. The system requires neither restriction enzyme digestion nor allele-specific oligonucleotides as conventionally applied for allele-specific hybridization of slot blots. This method clearly allows for the detection of the mutant allele by inspection, after agarose gel electrophoresis of a single PCR reaction. DNA from 40 patients with familial defective apo B-100 due to the G to A mutation at nucleotide 10,708 in the apo B gene and their normal relatives was analyzed. Complete agreement with allele-specific hybridization of slot blots confirms supposition that the system is effective to screen a larger population.     

The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability.

The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.     

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.     

Apolipoprotein E-4Philadelphia (Glu13----Lys,Arg145----Cys). Homozygosity for two rare point mutations in the apolipoprotein E gene combined with severe type III hyperlipoproteinemia.

The molecular defect in a 24-year-old white female with severe type III hyperlipoproteinemia has been elucidated. The patient's apolipoprotein (apo) E migrated in the apoE-4 position on isoelectric focusing gels. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apoE-4 variant had a smaller apparent molecular weight than apoE-4(Cys112----Arg). Sequence analysis of DNA amplified with the polymerase chain reaction revealed two nucleotide substitutions in the proband's apoE gene. A C to T mutation converted arginine (CGT) at position 145 of the mature protein to cysteine (TGT) thus creating the apoE-2 variant. A second G to A substitution at amino acid 13 led to the exchange of lysine (AAG) for glutamic acid (GAG), thereby adding 2 positive charge units to the protein and producing the apoE-5 variant. Computer analysis of the apoE-4Philadelphia gene revealed that the G to A mutation in exon 3 resulted in the loss of an AvaI restriction enzyme site. The second mutation, a C to T substitution in the fourth exon of the apoE gene, eliminated a cleavage site for the enzyme BbvI. Using these restriction fragment length polymorphisms as well as DNA sequence analysis we have demonstrated that the patient is homozygous for both point mutations in the apoE gene.     

A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease.     

Heterozygosity for apolipoprotein E-4Philadelphia(Glu13----Lys, Arg145----Cys) is associated with incomplete dominance of type III hyperlipoproteinemia.

Apolipoprotein (apo) E-4Philadelphia is a double mutant of apoE in which residue 13 of the mature protein, glutamic acid (GAG), is replaced by lysine (AAG) and amino acid 145, arginine (CGT), is converted to cysteine (TGT). These mutations result in two restriction fragment length polymorphisms for the enzymes AvaI and BbvI, a smaller apparent molecular weight of apoE-4Philadelphia on sodium dodecyl polyacrylamide gels, and severe type III hyperlipoproteinemia (HLP) in a 24-year-old homozygous female (Lohse, P., Mann, W. A., Stein, E. A., and Brewer, H. B., Jr. (1991) J. Biol. Chem. 266, 10479-10484). In the current study, we have extended our analysis to include nine additional family members of the Philadelphia kindred spanning four generations. DNA and protein analysis demonstrated that the originally described propositus is a true homozygote for the epsilon-4Philadelphia allele and that six of the nine family members are heterozygous for the mutated allele and the normal epsilon-3 allele or, in one case, the epsilon-4 allele. Heterozygosity for apoE-4Philadelphia leads to the expression of a moderate form of type III HLP without clinical manifestations. These results are consistent with a dominant mode of inheritance of this dyslipoproteinemia. The simultaneous presence of unaffected individuals, heterozygotes, and a homozygote in the Philadelphia kindred makes it possible for the first time to demonstrate that the mutant apoE exhibits an incomplete or partial dominance of type III HLP. Heterozygosity for the normal epsilon-3 allele appears to have an influence on the expression of type III HLP, resulting in a phenotype intermediate between that of the two homozygous states.     

Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors.

Apo E5(Glu3----Lys) is a naturally occurring apolipoprotein E (apo E) mutant found in patients with hyperlipoproteinemia and atherosclerosis. It has been shown to have a high affinity for low density lipoprotein (LDL) receptors. In this study, mutant apo E5 was produced by Chinese hamster ovary cells by means of an in vitro site-directed mutagenesis technique, and its LDL receptor binding activity was assessed. The apo E5 obtained from gene expression bound more readily to the LDL receptor than did plasma apo E3. The concentrations required for 50% competitive binding of 125I-labeled LDL to the LDL receptors were 58.9 ng/ml for plasma apo E3 and 25.7 ng/ml for the expressed apo E5. The expressed apo E5 displayed 229% normal binding. This result is highly consistent with that obtained with plasma apo E5, which showed 217% normal binding. Although the experimental apo E isoproteins contained more sialic acid than plasma apo E, the extent of sialylation had no effect on the receptor binding of apo E.     

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.     

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.     

Apolipoprotein E phenotypes in patients with myocardial infarction

Summary The frequencies of genetic apo E isoforms E2, E3 and E4 were determined in 523 patients with myocardial infarction and compared to those in a control group (1031 blood donors). A significant difference in the frequency of apo E4 was noted between patients and controls (0.05> P>0.025). No differences in the frequencies of isoforms E3 and E2 were observed. In particular, there was no significant difference between the two groups in the frequency of apo E2 homozygosity. a condition that is associated with type III hyperlipoproteinemia. However, all E2 homozygote survivors of myocardial infarction had hyperlipoproteinemia type III (cholesterol 269±29 mg/dl; triglyceride 419±150 mg/dl; age 54±14 years; N=5). On the contrary, E2 homozygote controls (all apo E-2/2 blood donors and their apo E-2/2 relatives who were from the same age range as the patients) had primary dysbetalipoproteinemia but normal or subnormal plasma cholesterol concentrations (cholesterol 184±28 mg/dl; triglyceride 151±52 mg/dl; age 56±13 years; N=11). This indicates that E2 homozygotes with hyperlipoproteinemia type III who occur rarely in the population but comprise about 1% of myocardial infarction patients have a markedly increase risk for coronary atherosclerosis, whereas the risk for E2 homozygotes with normal or subnormal plasma cholesterol (=primary dysbetalipoproteinemia) may be considerably lower than for the general population. The data illustrate the complex relationship between apo E genes, lipid levels, and risk for atherosclerosis.