Identification of a new apolipoprotein E variant (E2 Arg142 → Leu) in type III hyperlipidemia

ATHEROSCLEROSIS Atherosclerosis I12 (1995) 19-28

Identification of a new apolipoprotein E variant @2 Arg14,- Leu) in type III hyperlipidemia Pascale Richard”, Maria Pascual de Zulueta”, Isabel Beuclerb, Jean-Luc De Gennesc, AndrC Cassaigne”, Albert Iron*a “Dipartement

de Biochimie MPdicale et Biologie Mokculaire, Universirk de Bordeaux 2, 3.3076 Bordeaux Cede.y, France bLaboratoire de Biochimie des Lipides, H6pital de la Pitik. 75651 Paris Cedex 1.3, France ‘Clinique des Maladies Mc!taboliques, CHU Piti&Salpttri>re, 75013 Paris, France

Received 3 December 1993; revision received 1 July 1994;accepted 13 July 1994

Abstract A new rare apolipoprotein E mutant was identified as we were investigating the apolipoprotein E genotype of patients with type III hyperlipidemia (HLP III). The unusual DNA restriction fragment length polymorphism profile

and then the sequenceanalysis of a PCR amplified fragment of the proband’s apo E gene revealeda simple basesubstitution (G-T) at nucleotide 3836. This mutation leads to the replacement of arginine by leucine at position 142of the mature protein. The proband carried the mutant allele at the heterozygous status with an ~3allele. Subsequently, analysis of the proband’s father’s apo E gene showed the samemutated allele associatedwith an ~2 allele. The two subjects presented a dysbetalipoproteinemia in which this new apo E variant could be implicated. Keywords:

Type III hyperlipidemia; Apolipoprotein E variant; Restriction fragment length polymorphism; DNA

sequencing

1. Introduction Apolipoprotein (apo) E is a 299 amino acid glycoprotein of 34.2 kDa [ 11present in several plasma lipoprotein fractions, including chylomicrons, very low density lipoproteins (VLDL) and their remnants, and some subfractions of high density lipoproteins (HDL) [2]. The major physiological role of apo E consists of mediating the interaction of li* Correziponding author.

poproteins with receptors, including the cell surface LDL receptor and the chylomicron remnant receptor [3]. Since it serves as a ligand for these receptors, apo E plays a central role in determining the metabolic fate of plasma lipoproteins and therefore the plasma levels of cholesterol and triglycerides [4]. The human apo E gene is located on chromosome 19 and spans on 3.7 kb including four exons. Genetic variations at the apo E gene locus underlie the protein polymorphism, giving rise to three

002l-9150/95/909.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9 150(94)05393-W

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P. Richard et al. /Atherosclerosis

common alleles: ~2,~3,~4.This polymorphism is in the fourth exon. At the protein level, it corresponds to the presenceof Arg (codon CGC) or Cys (codon TGC) in positions 112 and 158, leading to Cysi i2/Argr5sin E3, Arg, 12/Arg158 in E4 and Cysr&ysr5s in E2 [5]. The ~3 allele frequency is the highest in the population; so apo E3 is considered to be the wild isoprotein. The three major apo E alleles and their resulting protein products have a noticeable impact on the variations of plasma lipid and lipoprotein levels. Compared with ~3 allele, ~2 allele is associated with lower mean values of total cholesterol, LDL cholesterol and LDL apo B, and with higher levels of triglycerides. In contrast, ~4 allele has the opposite effect on all these plasma parameters [6]. The amino terminal domain - approximately between residues 140 and 160 - is a sequenceenriched in basic amino acids that can interact with the acidic amino acids of the ligand binding domain of the LDL receptor [7]. Isoforms E3 and E4 normally bind to the LDL receptor, but isoform E2 performs less than 2% of normal binding [8]. Since apo E2 differs from apo E3 by a single amino acid substitution at residue 158,this single change is responsiblefor the binding defect. Homozygosity for apo E2 predisposesto the development of a type III hyperlipoproteinemia due to a delayed metabolic clearance of apo E-containing lipoparticles. In addition to the three common apo E isoforms, several rare variants have been identified, the majority of them being associatedwith perturbations of lipid metabolism. Most of these mutations are located in the domain involved in the cellular receptor binding [9]. An earlier study demonstrated that a human apo E variant with the replacementof arginine at position 142by cysteine may be associated with a dominant transmission of type III hyperlipoproteinemia [ 101.The expression of this variant in E. coli and binding studies with LDL receptors showed that the single cysteine for arginine change at position 142 reduces the receptor binding to about 20% in comparison with apo E3 [l 11. The characterization of apo E isoproteins can be carried out by the phenotyping of plasma proteins using isoelectric focusing (IEF) on the basis of the global charge of the protein [12]; the relative

II2 (1995) 19-28

charges are zero, +l and -1 for apo E3, apo E4 and apo E2, respectively. Molecular biology techniquesallow the direct detection of the nucleotide substitution using a PCR amplification followed by a hybridization with allele-specific oligonucleotide probes (ASO) [ 131.HhaI restriction fragment length polymorphism (RFLP) analysis of a PCR amplified domain of the apo E gene [14] permits the detection of the three common alleles and of several rare mutations [15]. The identification of e2(G1yrZ7-Asp), also called Weisgraber allele, is possible after Taq I digestion [16]. We report here the identification of a fragment with an abnormal size revealed by RFLP genotyping studies of subjectswith type III hyperlipoproteinemia. This fragment was present in two related subjects, the proband and his father. We showed that it corresponds to an undescribed apo E mutation at codon 142 (Leu for Arg) characterized at the gene level. 2. Materials and methods 2.1. Subjects and blood samples

The proband (C.S.) was a patient with type III hyperlipoproteinemia according to the classic criteria for HLP III (hypercholesterolemia, hypertriglyceridemia, VLDL cholesterol and presenceof a broad fl fraction). The proband’s father (R.S.) was studied,subsequently. Blood was collected after an overnight fast and the serum was separatedby centrifugation for lipid and lipoprotein analysis. Potassium-EDTA anticoagulated blood samples were also collected. The sampleswere centrifuged for 10 min at 500 x g to separate plasma from cells. Genomic DNA was isolated from leukocytes by a standard method [17]. 2.2. Serum lipids and apolipoproteins

Triglycerides, total cholesterol, and HDL cholesterol were determined by standard enzymatic techniques. Apolipoproteins A-I and B levels were measured by immunonephelemetry on a BNA analyser (Behring, Marburg, Germany). 2.3. Lipoprotein electrophoresis

Electrophoresis was performed on agarose gels

P. Richard

et al. /Atherosclerosis

in barbital buffer, pH 8.6. Electrophoresis of serumwas carried out for 30 min at 100V, then the gels were fixed and stained. The electrophoretic strips were scanned with a densitometer. 2.4. Apolipoprotein E phenotyping IDL were isolated from the plasma of the subjects, who had fasted, at density d < 1.006 in a 2 h spin in an NVT rotor on a Beckman Optima XL90 ultracentrifuge at 90 000 rev./min according to [18]. Lipoproteins were delipidated with ethanol/ethyl ether and solubilized with 8.6 M urea and 1%dithiothreitol (DTT) [ 191.Isoelectrofocusing was carried out in a 7.5% acrylamide gel containing pH 4-6 ampholines [19]. Two-dimensional electrophoresis was performed in two steps:first an electrofocusing was carried out as described, and then a second electrophoresis was run perpendicularly in a 15% acrylamide gel with 0.1% sodium dodecyl sulphate (SDS) [20]. Proteins were revealedwith Coomassie

112 (1995)

Blue and compared with a normal control sample run in the same conditions. 2.5. Apolipoprotein E genotyping Apo E genotyping was carried out with a procedure described elsewhere [ 151.In brief, a 292 bp DNA fragment was PCR amplified with two primers, Pl (5’AA CAA CTG ACC CCG GTG GCG3’) and P2 (5’AT GGC GCT GAG GCC GCG CTC3 ‘). An aliquot was digested with HhaI endonuclease(Gibco BRL, PaisIey, UK) for 2 h at 37”C, and another aliquot with TuqI enzyme (Gibco) for 2 h at 65°C. The restriction fragments were separated by electrophoresis on a loo/ polyacrylamide gel as well as the DNA size marker pBR322 DNA HaeIII digested (Marker V, Boehringer, Mannheim, Germany), and then revealed by staining with ethidium bromide, viewed under UV illumination and finally photographed. A schematic representation of the expected restriction fragments for ~3, ~2, ~4, and ~2(Gly,~,-Asp) alleles is shown in Fig. 1. EXON 4

ApoEgene

.

/

/H

.

.

.

\

1

61

1 161

91

1

61

J. J. 1 161

91

[

61

L

fragments

\

292

I’

139

1

4, J I J.

k

1 161

3’

\

,/p1-

/

k J.

Taql

P2

dl A’+

5.1

PCR product

21

19-28

J. 48 I

4J.r I I I 1 E3

allele

k.LJ.

J. 83

& 91

35

101

153

1 1 E2 allele .LJJ. I

1 &2[Argce~Lea]

1 &2[Glyl27->hp]

at~de

8bie

Fig. I. Schemeof restriction profiles of apo E PCR products: Hhal fragments for 64, 63, c 2. c2(Arg,42-Leu) alleles, and TQ~I fragments for e2(Gly12yAsp) (i.e. Weisgraber allele). Single arrow, t/ha1 cutting sites; double arrow, Taql cutting site. The length of each fragment generated by endonucleasedigestion is indicated in base pairs. The circled numbers representthe position of the amino acid change in the protein.

P. Richard et al. / Atherosclerosis I I2 (1995) 19-28

22

coronary or peripherical vascular disease (no extravascular cholesterol deposits, normal Doppler ultrasonography of lower limbs and carotids). He was taking a continuous treatment of ciprofibrate (DCI) which lowered his cholesterol and triglyceride values (Table 1). The proband’s father (R.S.) was a 75-year-old man. Physical examination revealed evidence of atherosclerosis with lower limb arteritis and carotid stenosis,and xanthomas involving elbows, kneesand arcus corneae. The plasma lipids and lipoprotein values are given in Table 1. The dyslipoproteinemia was successfully corrected with fibrate treatment.

2.6. DNA sequence analysis

The samesequenceof exon 4 of the apo E gene was amplified with PI and P2 primers and purified by agarose gel electrophoresis. The amplified DNA was then treated with 1 U/p1 of nuclease ‘Mung bean’ (Boehringer) for 10 min at 37°C to generateblunt ends for ligation. The pBluescript II KS phagemid (Stratagene, La Jolla, CA) was EcoRV cleaved and the apo E DNA fragment was cloned using 40 U/ccl of T4 DNA ligase (New England BioLabs, Beverly, MA) at 14°C overnight. The recombinant plasmid was used to transform Epicurian Coli XL1 Blue supercompetent cells (Stratagene, La Jolla, CA). The purified plasmid was sequenced according to Sanger’s method [21] with the T7 sequencing kit (Pharmacia Biotech Inc., Piscataway, NJ) using (a-32P)dATP. The primers used for sequencing were KS (5’CGAGGTCGACGGTATCG3’) and SK (5 ‘TCTAGAACTAGTGGATC3 ‘) for the downstream and upstream sequences,respectively.

3.2. Isoelectrofocusing

The proband’s apo E phenotyping revealed the presenceof two isoproteins, one with a normal E3 mobility and its sialylated forms and another with an abnormal mobility (charge -2) similar to a sialylated form of apo E2 (Fig. 2). 3.3. Two-dimensional electrophoresis

A two-dimensional electrophoresis showed two isoforms with their monosialylated and bisialylated forms: a normal apo E3 and an uncommon apo E2 whose migration corresponds to a more acidic protein with a lower molecular weight (Fig. 3).

3. Results 3.1. Characterization of patients

The proband (C.S.) was a 5 1-year-old Caucasian male who presented elevated levels of serum cholesterol and triglycerides. He had no symptoms of

Table 1 Serum lipid and apolipoprotein of IO values)

(C.S.) and of his father

Triglycerides (mgdl) Total cholesterol (mg/dl) LDL cholesterol (mgdl) HDL cholesterol (mgdl) Apolipoprotein A-I (mgdl) Apolipoprotein B (mg/dl) Total chol./triglycerides Total chol./HDL chol. B

(RX)

before

and after ciprofibrate

treatment

Father

Proband

Treatment

Apo A-l/Ape

levels of the proband

Before

After

Before

After

233 284 193 44 146 153 2.8 6.5

98 I98 I28 50 I64 99 4.6 4.0 1.7

259 394 293 49 170 140 3.4 8.1 1.2

155 275 I85 59 I80 109 4.0 4.6 1.7

I.0

(means

P. Richard et al. /Atherosclerosis

23

112 (I 995) 19-28

E4 E3 E2

4-

pro ApoAl

1-

ApoAl

Fig. 2. Determination of the proband’s apo E phenotype by isoelectric focusing of lip6proteins isolated by ultracentrifugation. Lane I: E3/E2 control obtained from VLDL lipoprotein fraction. Lane 2: E4/E3 control from VLDL lipoprotein fraction. Lane 3: IDL lipoprotein fraction from the proband, C.S. Lane 4: position of pro apo AI and apo AI proteins on HDL fraction.

3.4. Restriction analysis

fragment

length polymorphism

The genotyping of the proband DNA yielded restriction fragments that were consistent with the presence of an ~3 allele (i.e. corresponding to Cyst,, and Argtss) associated with an unusual larger fragment of approximately 100 base pairs. The apo E genewas also screenedfor the presence of a Weisgraber allele: no cleavage with 7’aqI was observed (Fig. 4). The genotyping of the proband’s father’s DNA revealed that he also carried the abnormal HhaI restriction pattern associated with an 62 allele (Fig. 4).

3.5. DNA sequence analysis

Ampicillin resistant clones were first screened using a PCR amplification of recombinant plasmidic DNA and cleavage of PCR products with HhaI endonuclease restriction enzyme. We obtained the restriction profile of the mutant allele at the homozygous status (data not shown). Then, three independent clones were sequenced. The sequencing of the proband’s apo E allele identified a point mutation at codon 142; the normal CGC sequence [22] was changed to CTC which eliminates the usual HhaI cleavagesite (Fig. 5). Codons 112 and 158 were TGC (Cys), according to an 62 allele. The abnormal fragment could

24

P. Richard et al. /Atherosclerosis

112 (1995) 19-28

Ex(Zs)

Fig. 3. Two-dimensional schematic representation isoform).

polyacrylamide gel electrophoresis of apolipoprotein of spots (apolipoproteins E and their monosialylated

have resulted from the loss of an HhaI site of a parental ~2 allele, giving rise to a 101 base pair fragment in addition to typical e2 restriction fragments. The sequencing of the proband’s father’s apo E DNA revealed the presenceof the samemutation at codon 142. 4. Discussion Type III hyperlipoproteinemia is usually in-

E3/E2(Arg1,2-Leu) (s) and bisialylated

from the proband with a (2s) forms; Ex, unexpected

herited as a recessivetrait and is commonly associated with an E2lE2 phenotype. We identified a new apo E variant at the heterozygous status in two patients presenting a type III hyperlipoproteinemia. Our data demonstrate that restriction genotyping can bring important information when it is used for the study of HLP III and apo E gene variations. Structural changes at the DNA level can alter the electrophoretic behaviour of the protein apo E, therefore the separation of apo E isoforms, on the basis of their charge, may not be

P. Richard et al. /Atherosclerosis

25

I12 (199s) 19-28

::a92

‘883

:;I 01 31

61

35

Fig. 4. Restriction fragments observed following polyacrylamide gel electrophoresis of apo E PCR products. Lane pBR322/HrreIII: DNA molecular weight marker. Lanes PCR: uncleaved amplilied DNA from the proband’s father c2le.2 (Arg142- Leu) and the proDNA endonuclease digestions. band ~3162 (Arg142-Leu). Lanes MaI and Taql: fragments resulting from amplified

suffkient to identify a precise phenotype. Restriction isotyping may provide a more accurate determination of apo E genotype by reducing the artefacts inherent to protein-based methods. DNA sequenceanalysis revealed that the new variant was the result of a G to T mutation at nucleotide 3836 of the common ~2 allele, converting Arg (CGC) to Leu (CTC) at position 142, thus creating a protein with an electrophoretic mobility corresponding to an apo E with a charge of -2.

GC rich regions are potentially ‘hot spots’ for DNA mutations [23], and the use of HhaI or its isochizomers (recognition site: GCGC) may provide opportunities to detect these mutations. The technique based on HhaI restriction profile analysis is more accurate than PCR amplification and hybridization with allele-specific oligonucleotides (testing only the 112 and 158 codons) which identified the proband as an ~31~2heterozygote (data not shown). The replacement of Arg by Leu in a

26

P. Richard et al. /Atherosclerosis

ATC -

RIWAN’I’

NORMAI,

G F--

:

I

Leu 141 Leu

; I CT

c A His 140 His c I :: T I Ser 139

Ser

:: Ala 138 _<; I

Ala

Fig. 5. Sequenceof the relevant part of the proband mutant e2[Arg142-Leu] allele. The box indicates the codon with the G-T mutation leading to ArgId2-Leu substitution. The corresponding sequenceof the normal allele is derived from [22].

Cysi iz/Argiss isoprotein is consistent with the bidimensional electrophoresis pattern that demonstrated a more acidic form with a lower molecular weight compared with a usual ~2 protein, These data illustrate the complementarity of phenotyping and genotyping procedures for detection of apo E mutations in HLP III. Binding studies of apo E variants indicate that the region of apo E implicated in the LDL receptor interaction is located in the centre of the protein, around residues 140- 160.There is no difference in

I I2 (1995) 19-28

receptor binding between apo E3 (CysIIZ/Arg1s8) and apo E4 (Arg1i2/Arg1ss);the defective receptor binding is entirely due to the substitution of Cys for Arg at position 158 corresponding to the E2 isoform (Cy~i~&ys~~s) [8]. Using site-specific mutagenesis,someauthors 1241have reported the existence of two apo E variants presenting lower binding activity: one with a substitution of alanine for lysine at residue 143and the other with proline for leucine at residue 144. Construction of other variants indicates that the receptor binding defect in this generegion is mainly due to the presenceof a mutation at position 142. This position seemsto be very important in the apolipoprotein-receptor interaction; the replacementof arginine 142by leutine appears to give rise to defective binding by disrupting the direct interaction of apo E with the LDL receptor. A human variant with a cysteine substituted for arginine at residue 142 is also known to be associatedwith a reduction of several interactions: apo E binding to LDL receptors, affinity for the monoclonal antibody 1D7, affinity for heparin [9]. Thesedata suggestthat residues 142to 144 are especially crucial for binding to the LDL receptor. The relation between the E2(Arg,,-Leu) isoform and hyperlipidemia is unclear. It has been previously reported that the degree of the binding defect caused by various mutations of apo E does not correlate with the severity of the observed hyperlipidemia [25]. Most of the rare mutations of the apolipoprotein E associated with HLP III presented a better binding affinity than the usual E2, which binds with l%-2% efficiency compared with the normal E3. Apo E isolated from e2/~2 subjects presents a defective binding to the LDL receptors in vitro regardlessof their serum cholesterol levels [26]. Differences in genetic, environmental and age factors may contribute to the variable expression of the disorder; these factors must be present in addition to ~2homozygosity for the full phenotypic expression of HLP III. It is not surprising that the proband’s father, heterozygotee2/e2(Arg142-Leu), has a severetype III hyperlipidemia with obvious clinical signs, frequently observed in patients homozygous for the e2 allele. The proband, heterozygote 63162 (Arg,,,-Leu), has a hyperlipidemic pattern. In

P. Richard

et al. / Alherosclerosis

general, heterozgous patients carrying the normal ~3 allele do not present lipid and lipoprotein abnormalities. The presence of the (Arg142-Leu) mutation may play an important part in the expression of type III hyperlipidemia. In these two patients, biological parameters were successfullycorrected under fibrate treatment (fenofibrate, 400 mg b.d; replaced since 1986 by ciprotibrate, 200 mg b.d.). Fibrates are often considered as selected drugs for therapy of patients with type III hyperlipidemia [27,28]. A good responseto treatment indicated that the presenceof the e2(Arg142-Leu) allele did not render the patients resistant to tibrates. In conclusion, the detection of a new apo E variant in two related patients provides a good opportunity for the study of this apolipoprotein and can help the development of in vitro studies of recombinant apo E variants created by site-directed mutagenesis.

References [I] Rail, S.C., Jr., Weisgraber, K.H. and Mahley, R.W., Human apolipoprotein E: the complete amino acid sequence, J. Biol. Chem., 257 (1982) 4171. [2] Mahley, R.W., Innerarity, T.L., Rail, SC., Jr. and Weisgraber, K.H., Plasma lipoproteins: apolipoprotein E structure and function, J. Lipid Res., 25 (1984) 1277. [3] Mahley. R.W. and Innerarity, T.L., Lipoprotein receptors and cholesterol homeostasis, Biochim. Biophys. Acta, 737 (1983) 197. [4] Havel, R.J., Yamada, N. and Shames, D.M., Role of apolipoprotein E in lipoproteins metabolism. Am. Heart J., I I3 (1987) 470. [5] Weisgraber, K.H., Rail, SC., Jr. and Mahley, R.W., Human apoprotein E heterogeneity: cysteine-arginine interchangesin the amino acid sequenceof the apo E isoforms, J. Biol. Chem., 256 (1981) 9077. [6] Sing, C.F. and Davignon, J., Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation, Am. J. Hum. Genet., 37 (1985)268. [7] Innerarity, T.L., Friedlander, E.J., Rail, S.C., Jr., Weisgraber, K.H. and Mahley, R.W., The receptor binding domain of human apolipoprotein E: binding of apolipoprotein fragments, J. Biol. Chem., 258 (1983) I2 341. [8] Weisgraber, K.H., Innerarity, T.L. and Mahley. R.W.. Abnormal lipoprotein receptor-binding activity of human E apoprotein due to cysteine-arginine interchange at a single site, 1. Biol. Chem., 257 (1982) 2518. [9] Mahley, R.W., Innerarity. T.L.. Rail. SC., Jr.,

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Weisgraber, K.H. and Taylor, J.M., Apolipoprotein E: genetic variants provide insights into its structure and function, Curr. Opin. Lipidol., i (1990) 87. 1101Rail, S.C., Jr., Newhouse, Y.M., Clarke, H.R.G. et al., Type III hyperlipoproteinemia associated with apolipoprotein E phenotype E3/3: structure and genetics of an apolipoprotein E3 variant, J. Clin. Invest., 83 (1989) 1095. VII Horie, Y ,, Fazio, S., Westerlund, J.R., Weisgraber, K.H. and Rail, S.C., Jr., The functional characteristics of a human apolipoprotein E variant (cysteine at residue 142) may explain its association with dominant expression of type III hyperlipoproteinemia. J. Biol. Chem., 267 (1992) 1962. 1121Luley, C., Prellwitz, W., Oster, 0. and Kloer, H.U., Determination of apolipoprotein variants by isoelectric focusing in agarose, Anal. B&hem., 163 (1987) 182. [I31 Main, B.F., Jones,P.H., McGillivray, R.T. and Banfield, D.K., Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers, J. Lipid Res., 29 (1991) 183. (141 Hixson, J.E. and Vernier, D.T., Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hhal, J. Lipid Res., 31 (1990) 545. 1151 Richard, P., Thomas, G., Pascual de Zulueta. M. et al., Determination of common and rare genotypes of human apolipoprotein E by specific restriction profiles of polymerase chain reaction amplified DNA, Clin. Chem., 40 (1994) 24. 1161Kontula, K., Aalto-Setala, K., Kuusi T., Hamllainen. L. and Syvinen, A.C., Apolipoprotein E polymorphism determined by restriction enzyme analysis of DNA amplified by polymerasechain reaction: convenient alternative to phenotyping by isoelectric focusing, Clin. Chem., 36 (1990) 2087. 1171 Miller, S.A., Dykes, D.D. and Polesky, H.F., A simple salting-out procedure for extracting DNA from human nucleated cells, Nucleic Acid Res., I6 (1988) I2 15. 1181Havel, R.J.. Eder, H.A. and Bragdon, J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest.. 34 (1955) 1345. [I91 Bouthillier, D., Sing, F. and Davignon, J., Apolipoprotein E phenotyping with a single gel method: application to the study of informative matings. J. Lipid Res., 24 (1983) 1060. 1201Giancarlo, G., Gregg, R.E. and Brewer. H.B., Jr.. Apolipoprotein E Bethesda isolation and partial characterisation of a variant of human apolipoprotein E isolated from very low density lipoproteins, Biochim. Biophys. Acta. 794 (1984) 333. 1211Sanger. F., Nicklen, S. and Coulson, A.R.. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74 (1977) 5463. WI Paik, Y.K.. Chang, D.G., Reardon. C.A.. Davies, C.E., Mahley. R.W. and Taylor, J.M., Nucleotide sequence and structure of the human apolipoprotein E gene, Proc. Natl. Acad. Sci. USA. 82 (1985) 3445.

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(231 Barker, D., Schafer, M. and White, R., Restriction sites containing CpG show a higher frequency of polymorphism in human DNA, Cell, 36 (1984) I3 I. (241 Lalazar, A., Weisgraber, K.H. and Rail, SC., Jr., Site specific mutagenesisof human apo E. Receptor binding activity of variants with single amino acid substitutions, J. Biol. Chem., 263 (1988) 3542. [25] Chappel, D.A., High receptor binding affinity of lipoproteins in atypical dysbetalipoproteinemia (type III hyperlipoproteinemia), J. Clin. Invest., 84 (1988) 1906. [26] Rail, SC., Jr., Weisgraber, K.H., Innerarity, T.L. and

Mahley, R.W., Identical structural and receptor binding defects in apolipoprotein E2 in hypo-, normo-, and hypercholesterolemic dysbetalipoproteinemia, J. Clin. Invest., 71 (1983) 1023. [27] Kuo, P.T., Wilson, A.C., Kostis, J.B., Moreyra, A.B. and Dodge, H.T., Treatment of type HI hyperlipoproteinemia with gemlibrozil to retard progression of coronary heart disease,Am. Heart J., 116 (1988) 85. [28] O’Connor, P., Feely, J. and Shepherd, J., Lipid lowering drugs, Br. Med. J., 300 (1990) 667.