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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:865-872.)
© 1997 American Heart Association, Inc.

Articles

The Apolipoprotein E2(Arg145Cys) Mutation Causes Autosomal Dominant Type III Hyperlipoproteinemia With Incomplete Penetrance

Willem J.S. de Villiers; Deneys R. van der Westhuyzen; Gerhard A. Coetzee; Howard E. Henderson; ; A. David Marais

From the MRC/UCT Research Unit for the Cell Biology of Atherosclerosis, Department of Medical Biochemistry (W.J.S. de V., D.R. van der W., G.A.C.), and the Department of Chemical Pathology and Medicine (H.E.H., A.D.M.), University of Cape Town Medical School, Cape Town, South Africa.

Correspondence to Dr A. David Marais, Department of Medicine, University of Cape Town Medical School, Observatory 7925, South Africa. E-mail dmarais{at}uctgsh1.uct.ac.za

	Abstract

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Abstract Type III hyperlipoproteinemia (type III HLP) is an atherogenic disorder of lipoprotein metabolism characterized by the accumulation of cholesterol-enriched VLDL and is usually associated with homozygosity for a normal variant of apoE, apoE2. ApoE2(Arg145Cys) is a rare variant arising from a CT transition at nucleotide 4031 and has been linked to type III HLP. Ten subjects from a group of 42 unrelated individuals with proven type III HLP were found to be either heterozygous or homozygous for the apoE2(Arg145Cys) mutation by DNA sequencing. The apoE4-Philadelphia (Glu13Lys, Arg145Cys) variant was subsequently excluded. None of 4 homozygotes (3 blacks and 1 of mixed ancestry) developed ischemic heart disease, but they did present with xanthomata. In contrast, 6 heterozygous subjects presented mainly with ischemic heart disease but generally lacked physical signs. Cholesterol concentrations ranged from 6.2 mmol/L to 13.3 mmol/L and triglyceride levels from 3.2 to 13.2 mmol/L. The dyslipoproteinemia in homozygous and heterozygous subjects was indistinguishable. Family investigation identified an additional 10 heterozygous mutant-allele carriers, of whom 3 had type III HLP. This unique cohort of patients indicates that the apoE2(Arg145Cys) mutation is relatively common in several population groups in our region and may be particularly prevalent in blacks. There was no clear allele dosage effect present for the development of dyslipoproteinemia or atherosclerosis. The mode of inheritance is for the first time clearly established to be autosomal dominant with incomplete penetrance.

Key Words: type III hyperlipoproteinemia • autosomal dominant inheritance • apoE2(Arg145Cys) mutation • apolipoprotein E • dysbetalipoproteinemia

	Introduction

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ApoE is one of the major protein constituents of chylomicron and VLDL remnants and serves as a ligand for the receptor-mediated clearance of these lipoprotein particles by hepatic lipoprotein receptors.^{1 2} Type III HLP is a genetic disorder of lipoprotein metabolism in which defects in apoE binding lead to impaired clearance of remnant particles and increases in plasma concentrations of TC, TGs, and apoE. Cholesteryl ester enrichment of chylomicron and VLDL remnant (ie, ß-VLDL) particles predisposes affected individuals to the premature development of coronary and/or peripheral vascular atherosclerotic disease. The most distinctive feature of the disorder is the presence of lipid deposits, such as tuboeruptive cutaneous and tendinous xanthomata, as well as palmar crease xanthomas.³

Three common genetic variants of apoE were originally identified by isoelectric focusing and were accordingly named apoE2, apoE3, and apoE4.^{4 5} Molecular characterization has shown that the most common apoE3 isoform is encoded by the apoE3(Cys112, Arg158) allele. The common apoE2 and apoE4 isoforms are encoded by the (Arg158Cys) and (Cys112Arg) alleles, respectively, that differ from the apoE3 allele by single point mutations at codons 158 and 112, respectively.^{6 7} Homozygosity for the apoE2(Arg158Cys) allele is present in the vast majority of subjects with type III HLP. However, <5% of all apoE2(Arg158Cys) homozygotes in European populations develop type III HLP, presumably because additional defects causing either overproduction of TG-rich lipoproteins or additional decreases in VLDL or remnant clearance are required to develop the phenotype. Additional genetic, hormonal, and/or environmental factors may therefore contribute to disease expression, and diabetes mellitus, alcoholism, obesity, hypothyroidism, and renal failure have been noted to trigger the onset of type III HLP.⁸

The domain of apoE between amino acids 130 and 150 is responsible for the high-affinity binding to the LDL receptor^{9 10} ; this is mediated by direct ionic binding of some of the positively charged amino acid residues in the binding domain with negatively charged amino acid residues in the ligand-binding domain of the LDL receptor. ApoE variants with mutations that have removed a positively charged amino acid in the binding domain have been described in patients with type III HLP: apoE2-Christchurch (Arg136Ser),¹¹ apoE2-Heidelberg (Arg136Cys),¹² apoE3(Cys112Arg, Arg142Cys),^{13 14} apoE2(Arg142Leu),¹⁵ apoE1-Harrisburg (Lys146Glu),^{16 17} apoE2(Lys146Gln),¹⁸ apoE4-Philadelphia (Glu13Lys; Arg145Cys),¹⁹ and apoE2(Arg145Cys).^{20 21 22} Duplication of an amino acid sequence outside the binding domain in the apoE3-Leiden variant (Cys112Arg; residues 120-126 or 121-127) is also associated with type III HLP.²³

Interestingly, some of these rare apoE mutants, ie, apoE3(Cys112Arg, Arg142Cys), apoE1-Harrisburg, apoE2(Lys146Gln), apoE3-Leiden, and apoE4-Philadelphia, lead to type III HLP in the heterozygous state and are therefore inherited as dominant traits.^{14 16 24 25 26} The mode of inheritance and severity of disease expression do not, however, directly correlate with mutant apoE binding studies in vitro. LDL receptor–binding activity compared with wild-type apoE3 ranges between 20% for apoE3(Cys112Arg, Arg142Cys),²⁷ 25% for apoE3-Leiden,²⁸ and 40% for apoE2(Lys146Gln).²⁹ Binding to the LDL receptor–related protein for apoE1-Harrisburg was found to be only 18% of that of normal apoE3.³⁰ This apparent discrepancy between the magnitude of the in vitro binding defect and the occurrence of type III HLP remains a major unsolved question in apoE metabolism. In particular, it is unclear why the common apoE2(Arg158Cys) variant is not dominantly associated with severe type III HLP, as would be anticipated from its severe reduction (<2% of normal) of in vitro binding.²⁰

We found several subjects in our group of type III HLP patients who were not homozygous for the apoE2(Arg158Cys) allele; in addition, instances of autosomal dominant inheritance were evident. In this study we demonstrate that the rare apoE2(Arg145Cys) mutation is a common cause of type III HLP in our region and is associated with a dominant mode of inheritance with incomplete penetrance.

	Methods

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Subjects
Hyperlipidemic patients attending the Groote Schuur Hospital and the University of Cape Town Lipid Clinic were screened for biochemical and genetic parameters. Informed consent was obtained for all procedures. Investigations identified 42 unrelated subjects from various population groups with type III HLP; wherever possible, family members were also studied. Secondary causes of hyperlipidemia, such as diabetes mellitus, renal failure, and hypothyroidism were excluded by standard laboratory tests, except for 2 subjects (A.F. and G.B., Table 1) who were heterozygous for the apoE2(Arg145Cys) mutation and had diabetes mellitus. None of the subjects in the apoE2(Arg145Cys) group was overweight (BMI >32) or abused alcohol.

View this table: [in this window] [in a new window]   Table 1. Clinical and Biochemical Data at Presentation for Patients With Type III HLP Due toApoE2(Arg145Cys)

Lipid and Lipoprotein Analyses
Blood samples were collected into EDTA-containing tubes after an overnight fast. Plasma was analyzed after separation from cells by centrifugation at 500g for 10 minutes at room temperature. Plasma TC and TG concentrations were determined enzymatically (Boehringer Mannheim GmbH). HDL-C was assayed in the supernatant after precipitation of apoB-containing lipoproteins with either heparin/MnCl₂ or polyethylene glycol. Plasma apo(a) concentration was determined by a radioimmunoassay (Pharmacia) using the standards provided. Agarose electrophoresis for the detection of ß-VLDL was performed with the Beckman Paragon system (Beckman Instruments).

Type III HLP was diagnosed on the basis of the presence of (1) elevated plasma TC and TG levels, (2) a broad ß-band on agarose electrophoresis of whole plasma, and/or (3) a VLDL-C to plasma TG ratio >0.30 or a VLDL-C to VLDL-TG ratio >0.42 by mass.³ For isolation of VLDL (d<1.006 g/mL), plasma was adjusted to d=1.30 g/mL with KBr, layered beneath a "cushion" of saline/EDTA of d=1.006 g/mL, and centrifuged for 16 hours (Beckman SW40) at 100000g. The VLDL was recovered for lipid determinations, and lipid ratios were expressed in terms of mass. The VLDL supernatants were retained for denaturing gradient gel electrophoresis (SDS-PAGE) after delipidation.

ApoE Genotyping
Genomic DNA was isolated from leukocytes by the procedure of Parzer and Mannholter.³¹ ApoE genotyping of the common polymorphisms at codons 112 and 158 was performed by PCR of a 244-bp region encompassing both polymorphic sites, digestion of the PCR products with restriction enzyme Hha I, and electrophoresis on polyacrylamide gel as described previously.³²

Detection of the ApoE2(Arg145Cys) Mutation
Patients who were not homozygous for the apoE2(Arg158Cys) allele were analyzed further by sequence analysis of the amplified 244-bp apoE segment used for Hha I restriction isotyping, which was performed by direct sequencing with the Maxam-Gilbert method (DNA sequencing system kit, New England Nuclear)³³ after either repeated amplification using an end-labeled oligonucleotide primer or cycle sequencing using the fmol sequencing kit (Promega). Samples were electrophoresed through an 8 mol/L urea/6% polyacrylamide gel for 3 to 6 hours depending on the number of loadings. After the gels were dried, they were autoradiographed at room temperature for 48 to 60 hours with intensifying screens.

The apoE2(Arg145Cys) mutation was verified and detected further in subjects and family members by either RFLP analysis or rapid DNA sequencing. The mutation destroys an Fnu4HI restriction site, and therefore its presence was detected by digestion of the amplified apoE fragment with Fnu4HI. In brief, 10 µCi [-³²P]dCTP (Amersham) was added to the standard reaction mixture for apoE PCR amplification. The product was then electrophoresed on a 6% polyacrylamide gel, eluted, precipitated with ethanol, and dissolved in 10 µL water. Three units of Fnu4HI (New England Biolabs) and its specific buffer requirements were added to the amplified apoE sequence for digestion overnight at 37°C. Digested samples were loaded onto a 20% polyacrylamide nondenaturing gel and electrophoresed for 4 hours under constant current. The gels were dried and autoradiographed at room temperature for 24 hours with intensifying screens. Alternatively, unlabeled PCR product was digested with Fnu4HI and the fragment patterns distinguished by electrophoresis on standard sequencing gels with visualization by silver staining. Fnu4HI fragment sizes were estimated by comparison with pGEM DNA (Promega) size markers. Another approach was the rapid screening of family members by limited DNA sequencing of G and/or A nucleotides and electrophoresis on 8 mol/L urea/6% polyacrylamide gels.

Exclusion of the ApoE4-Philadelphia Mutation by RFLP Analysis
This procedure was performed as described previously.^{19 26} In brief, genomic DNA from the subjects was amplified by PCR using apoE-specific primers for exon 3. Enzymatically amplified DNA products were purified by electrophoresis on a 2% low-melting-point agarose gel and digested with restriction enzyme Ava I for 2 hours at 37°C under the conditions outlined by the manufacturer (New England Biolabs). Ava I restriction digests were analyzed on a 1.5% agarose gel and the fragments visualized by staining with ethidium bromide.

	Results

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Detection and Characterization of the ApoE2(Arg145Cys) Mutation
Twenty-six of the 42 subjects with ultracentrifugation-proved type III HLP were homozygous for the common apoE2(Arg158Cys) allele. Ten patients were homozygous for the apoE3 Hha I genotype, 4 were heterozygous for apoE3/apoE4(Cys112Arg), and 2 were heterozygous for apoE3/apoE2(Arg158Cys). It is important to bear in mind that apoE RFLP genotyping by Hha I demonstrates the common polymorphisms at codons 112 and 158 only; mutations at other sites may still affect the protein's migration pattern by isoelectric focusing. The apoE2(Arg145Cys) mutation was found in 10 subjects who had an apoE3 genotype. In these patients, analysis of the protein coding sequence of the apoE gene by DNA sequencing revealed a point mutation at nucleotide 4031 in exon 4. A CT substitution converted Arg, at residue 145 of the mature protein, encoded by CGT, to Cys encoded by TGT (Fig 1A). Of the remaining 6 patients who were not homozygous for the apoE2(Arg158Cys) allele, 1 was heterozygous for the apoE(Lys146Gln) mutation,³⁴ and the underlying cause in the others is still under investigation.

View larger version (125K): [in this window] [in a new window]   Figure 1. A, Partial DNA sequence of apoE, which indicates the GA transition at nucleotide 4031 or a CT transition on the complementary coding strand (Maxam-Gilbert chemical DNA sequencing). This results in an ArgCys amino acid change at residue 145. B, Detection of apoE2(Arg145Cys) by Fnu4HI restriction analysis. Electrophoretic separation of Fnu4HI fragments after digestion of apoE amplification products from DNA of a normal subject and a subject homozygous for the apoE2(Arg145Cys) mutation. [-32P]dCTP was added to the standard PCR reaction mixture. Fragment sizes (in bp) are shown to the left of the gel, and (-) and (+) represent undigested and digested samples, respectively.

Computer-assisted restriction mapping of the apoE2(Arg145Cys) gene sequence showed that this CT substitution abolished the restriction site for endonuclease Fnu4HI (5'-GCNGC-3'), where N may represent any nucleotide. The 244-bp wild-type amplified apoE sequence is normally digested by Fnu4HI into 9 fragments ranging from 9 to 66 bp. Loss of an Fnu4HI restriction site in the mutant allele results in a 42-bp fragment, thereby distinguishing it from the normal allele, which yields 2 fragments of 33 and 9 bp (Fig 1B). Both the 42- and 33-bp fragment sizes will be present in individuals heterozygous for the Arg145Cys mutation. To distinguish homozygotes from heterozygotes carrying the mutant allele, the mutant 42-bp fragment must be distinguished from a 40-bp fragment that is invariant. Although this distinction can be made when running the Fnu4HI-digested fragments on a 20% polyacrylamide gel (results not shown), the proximity of the two bands complicated the use of this assay for rapid pedigree and/or population screening. Alternative screening strategies were therefore employed for mutant-allele detection, and a more practical method proved to be the limited DNA sequencing of G and/or A nucleotides and electrophoresis on 8 mol/L urea/6% polyacrylamide gels. Fig 2 shows the A lanes from normal and heterozygous subjects. The characteristic triplet in the A lanes enabled this screening method to be employed to detect heterozygotes in family studies and also represents a useful modification for population prevalence detection of the mutant allele. Alternatively, screening by Bbv I RFLP analysis could be performed.²⁰

View larger version (44K): [in this window] [in a new window]   Figure 2. Detection of apoE2(Arg145Cys) by limited DNA sequencing. A lanes from normal (-) and heterozygous (+) subjects from the R. family are shown. The characteristic triplet in the A lanes enabled this screening method to be employed to detect heterozygotes in family studies.

It was also necessary to distinguish the apo E double mutant, apoE-4 Philadelphia, which had recently been identified¹⁹ and found to be associated with incomplete dominant expression of type III HLP.²⁶ This distinction was possible by RFLP analysis, as the GA substitution in codon 13 in exon 3 of the mutant apoE-4 Philadelphia allele results in loss of a restriction site for enzyme Ava I.²⁶ Ava I restriction digestion of PCR-amplified DNA confirmed the absence of this mutation from our cohort of subjects (data not shown). In addition, apoproteins prepared from the patients' VLDL fractions did not demonstrate the abnormal migration of apoE4-Philadelphia on SDS-PAGE that has been described.¹⁹

Clinical and Biochemical Data of Type III HLP Subjects With the ApoE2(Arg145Cys) Mutation
Of the 42 unrelated subjects with type III HLP genotyped at the apoE locus, 10 had the apoE2(Arg145Cys) mutation. Three black African subjects and 1 subject of mixed ancestry were homozygous for the mutation. Family investigation of 2 probands (R.M.-H. and M.R.) uncovered another 10 subjects who were heterozygous for the apoE2(Arg145Cys) mutation, of whom 3 (R.A., M.T., and M.K.) showed the type III HLP phenotype. The 3 African probands were unrelated, and none of their families could be characterized further. Apart from the 2 patients who had diabetes mellitus, secondary causes of type III HLP were absent.

The relevant clinical details of the patients who had type III HLP at presentation or identification are given in Table 1. The 3 unrelated homozygous black African patients (G.A., J.C., and S.D.) were Xhosa speaking and originated from the eastern Cape region of South Africa; they originally presented because of cutaneous xanthomata. The other patients of mixed or white race were detected because of the occurrence of IHD in a family member. Four patients had IHD, whereas palmar crease lipid deposition was present in 3 other individuals. None of the patients was being treated for a lipid disorder at presentation. TC levels ranged from moderately (6.2 mmol/L) to severely (13.3 mmol/L) raised; this wide range was mirrored by increases in TG levels (from 2.2 to 13.2 mmol/L). Plasma HDL-C concentrations were low in all cases. Apo(a) concentrations varied throughout the normal range for the population. Hha I RFLP analysis for the common polymorphisms at codons 112 and 158 showed the common apoE3 allele to be the second accompanying allele in all but 3 of the apoE2(Arg145Cys) heterozygous subjects. Two subjects had apoE4 and 1 had a common apoE2(Arg158Cys) variant as well as the apoE2(Arg145Cys) mutation.

Subjects in the R. family (for whom a pedigree is shown in Fig 3) were characterized further to determine the effect of the apoE2(Arg145Cys) allele on the expression pattern of type III HLP; these results are given in Table 2. TC and TG concentrations in the index case R.M.-H. and subject R.A. decreased significantly from presentation levels (Table 1) in response to diet and fibrate derivative therapy. Despite this improvement, VLDL particles remained relatively enriched with TC, thus retaining the typical type III HLP pattern. Young subjects heterozygous for the apoE2(Arg145Cys) allele did not display an abnormal lipoprotein phenotype, which suggests that expression of type III HLP in carriers may be age dependent. It is interesting to note that the heterozygous female subjects H.S. and Ra.R., at an age similar to the index case R.M.-H., were normolipidemic. Their VLDL-C to VLDL-TG ratios >0.3 do, however, indicate cholesteryl ester–enriched remnants and may signify a propensity to develop the type III HLP phenotype.

View larger version (21K): [in this window] [in a new window]   Figure 3. Family pedigree of the R. family. III-1 (M.-H.) is the index case. Squares represent males; circles, females; and diagonal lines, deceased subjects. Semishaded symbols represent heterozygotes for the apoE2(Arg145Cys) mutation. Initials refer to subjects whose data are shown in Table 2.

View this table: [in this window] [in a new window]   Table 2. Plasma Lipid and Lipoprotein Characterization in the R. Family With Type III HLP Due toApoE2(Arg145Cys)

	Discussion

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Type III HLP is generally associated with homozygosity for the common apoE variant apoE2(Arg158Cys). In this setting it is a multifactorial disease, since additional factors, genetic and/or environmental, are required for expression; age, sex, dietary fat, and obesity are known to influence disease manifestation.³ In our cohort of 42 proven type III HLP patients, the common apoE2(Arg158Cys) variant was detected in subjects of white, black, and mixed ancestry. In addition, 25% were found to be either heterozygous or homozygous for the rare apoE2(Arg145Cys) mutation. Genetic analyses were pivotal in this finding, as isoelectric focusing would have classified the homozygotes and the heterozygous patient (G.B.) with an additional apoE2(Arg158Cys) allele as having the common apoE2 protein isoform. Three unrelated black homozygotes for the apoE2(Arg145Cys) mutation presented with cutaneous signs while the fourth homozygote, of mixed ancestry, was discovered incidentally during investigation for hypertension. Three heterozygous index cases presented with IHD. Family investigations identified the type III HLP phenotype in 3 additional subjects, of whom 1 had premature IHD. Another 7 heterozygotes without the type III HLP phenotype were identified in the R. family; these subjects were all either relatively young and/or female.

Rall et al²⁰ reported homozygosity for the apoE2(Arg145Cys) allele in a 51-year-old African-American male diagnosed with type III HLP. Although the patient had cutaneous planar and tuberous xanthomas, there was no clinical evidence of atherosclerosis. In addition, 3 cases have been reported of the apoE2(Arg145Cys) mutation presenting in the heterozygous form. One was an African-American male who had typical type III HLP with coronary and cerebral atherosclerosis.²⁰ Of the 2 female subjects of unknown ancestry, 1 presented with type III HLP in adolescence²¹ while the other was normolipidemic with an abnormally cholesterol-enriched VLDL fraction.²² However, dominant or recessive inheritance could not be definitively established owing to the presence of the common apoE2(Arg158Cys) variant as the other apoE allele in each of these subjects. In the present cohort, the predominant second apoE allele was the "normal" apoE3 variant. Irrespective of this, type III HLP was dominantly inherited in apoE2(Arg145Cys) allele carriers, albeit with incomplete penetrance.

Five different apoE mutants associated with type III HLP have been reported to be autosomal dominant traits. The mutant apoE3(Cys112Arg; Arg142Cys) and apoE1-Harrisburg appear to be invariably associated with type III HLP.^{14 16 17 27} In the largest pedigree study published thus far, the apoE3-Leiden mutation (duplication of residues 120-126 or 121-127) was consistently associated with type III HLP in 42 heterozygotes.²⁵ Age and BMI correlated with disease expression, whereas sex had no influence on age of onset or biochemical and clinical severity. A pedigree study involving 40 heterozygotes with the apoE2(Lys146Gln) mutation showed a similar invariable association with type III HLP.³⁴ Phenotypic expression varied to a large extent, and sex contributed to the total variance in quantitative lipoprotein traits.

The apoE4-Philadelphia variant is associated with incomplete or partial dominant inheritance of type III HLP, and the presence of a normal apoE3 allele lessens disease severity in heterozygotes.^{19 26} The disease phenotype in the mutant-allele carriers seemed to be intermediate between the homozygous proband and unaffected siblings, suggesting an allele-dosage effect. In addition, the milder clinical presentation suggested that this mutant apolipoprotein could lie toward the less-severe end of dominant apoE mutants. Another naturally occurring apoE variant involving residue 145, apoE3-Kochi (Arg145His), is associated with type III HLP, but the pattern of inheritance could not be determined.³⁵

Mahley and coworkers^{9 36} initially suggested that the position of the apoE mutation determines the mode of inheritance of type III HLP. The loss of any positively charged amino acid within the 130-150–residue receptor-binding domain of the apoE molecule would compromise apoE binding by reducing ionic interactions with the ligand-binding domains of the LDL receptor and LDL receptor–related-protein, therefore resulting in "permanent" receptor-binding defects and a dominant mode of inheritance. These variants would be invariably associated with type III HLP and insensitive to secondary influences such as age, sex, BMI, and lipid composition of particles. In contrast, the apoE2(Arg158Cys) variant is located outside the receptor-binding domain and is associated with a low risk for developing type III HLP. The Cys residue at position 158 influences the conformation of the 130-150–residue -helical region and has a secondary effect on receptor-binding activity.^{36 37} The conformation of apoE2(Arg158Cys) is sensitive to its environment (such as diet-induced changes in lipid composition of lipoprotein particles), and this may explain the requirement of additional environmental and/or genetic factors for expression of type III HLP in apoE2(Arg158Cys) homozygosity.

Recent reports and evidence from our group of apoE2(Arg145Cys) mutants suggest that this original hypothesis needs modification. The apoE3-Leiden variant, situated outside the receptor-binding domain, disrupts the protein and is associated with dominant inheritance of type III HLP with high penetrance. However, secondary factors, such as age and BMI, do influence disease expression.²⁵ In apoE2(Lys146Gln) allele carriers, both sex and BMI contributed to a wide variation in phenotypic expression.³⁴

Several factors may account for the incomplete dominant penetrance displayed by the apoE2(Arg145Cys) mutation as described in this study. First, the LDL receptor–binding activity of the apoE2(Arg145Cys) protein is 45% that of apoE3, which is the closest to normal apoE3 binding of all the naturally occurring apoE variants described.²⁰ Second, plasma apoE levels in heterozygous subjects with apoE variants seem to correlate with penetrance of type III HLP expression, although a cause-effect relationship has not been established. ApoE4-Philadelphia allele carriers have no clinical manifestations and only moderately elevated plasma apoE levels.²⁶ By analogy, plasma apoE levels in "single"-substitution apoE(Arg145Cys) heterozygotes may be similarly moderately increased, indicating the less-severe nature of this dominant apoE variant. Third, the presence of normal apoE3 molecules on remnant particles may mediate their clearance more effectively. Fourth, although the outward-facing positively charged amino acid residues in the receptor-binding domain (residues 130-150) are essential in apoE and LDL receptor interaction, the side chain of Arg residue 145 points in a slightly different direction and lies at the edge of the electrostatic cloud that comprises the cluster.^{37 38} It may thus have a lesser role in ligand-receptor interaction. This may explain both the relatively mild decrease in LDL receptor binding of the apoE2(Arg145Cys) protein and why secondary influences like age and sex may contribute to incomplete or delayed penetrance of dominant inheritance.

The initial step in the clearance of apoE-enriched remnant lipoproteins is sequestration within the space of Disse in the liver by their binding to heparan sulfate proteoglycans.³⁹ All of the apoE variants associated with type III HLP are defective in binding to proteoglycans and in the cellular uptake initiated by heparan sulfate proteoglycans.^{30 39} The hierarchy of binding activity is apoE3>apoE2(Arg158Cys)>apoE2(Arg145Cys); both apoE3(Arg142Cys) and apoE3-Leiden display very little binding and uptake. This additional in vitro evidence of the less-severe nature of the apoE2(Arg145Cys) mutation may be another explanation for its incomplete dominant penetrance.

Premature onset of IHD was common among the group of heterozygotes. This mode of presentation differed from that of the Xhosa-speaking black homozygotes, who presented with cutaneous lipid deposits but no IHD. Although cutaneous signs were found more commonly in homozygotes, their hyperlipidemia was of similar severity to heterozygotes. The seemingly paradoxical resistance of homozygotes to atherosclerosis may be related to environmental or genetic factors. The traditional antiatherogenic life style of the rural African homozygous patients may have been protective against the earlier development of IHD. No allele-dosage effect was therefore evident, but it may have been obscured by differences in life styles of the probands.

In the case of individuals homozygous for the common apoE2(Arg158Cys) variant, a marked difference exists in the onset of expression of type III HLP between males and females, owing to differences in hormonal status.³ In men type III HLP is normally expressed between 30 and 40 years of age, whereas in women it occurs after menopause. In the R. family it may be significant that, of the carriers of the apoE2(Arg145Cys) allele >40 years, the men expressed overt type III HLP whereas the women did not. In a study of an extended multigeneration pedigree with the apoE3-Leiden mutation, type III HLP was dominantly inherited with a high rate of penetrance.²⁵ The second apoE allele affected expression of type III HLP: the E2 allele enhanced expression, as reflected by higher lipid and lipoprotein levels, and the E4 allele showed the opposite effect. In the currently described cohort of apoE2(Arg145Cys) subjects, most allele carriers were also heterozygous for the common apoE3 allele. The possible effects of accompanying apoE2 or apoE4 alleles on plasma lipid and lipoprotein levels can be assessed only in a larger cohort.

Given the diverse racial origin of the probands, it is probable that the apoE2(Arg145Cys) mutation is recurrent. This is not altogether surprising, as the CT nucleotide change at position 4031 is an example of a mutational "hot spot" in a CpG dinucleotide sequence.⁴⁰ Common ancestry for the three probands, given their close geographic proximity, could not be excluded, however, as sources used for genealogical studies such as parish archives and population and census records are unavailable and haplotype analysis of the apoE/C1/C2 gene cluster had not yet been done.³⁴ A high prevalence of this mutation in the black population is suggested by the presence of 3 unrelated Xhosa patients homozygous for the apoE2(Arg145Cys) mutation. This may have important implications for a population at increasing risk for IHD, owing to urbanization and adoption of a Westernized lifestyle. The prevalence of the apoE2(Arg145Cys) allele in the Xhosa-speaking African population of South Africa is unknown, although either RFLP analysis or limited A-lane DNA sequencing provides relatively convenient methods for mutation screening. Lipoprotein abnormalities in black South Africans are relatively rare,⁴¹ and <5% of a study population had elevated TC levels in the high-risk category, so identification of hyperlipidemia may be an effective initial strategy.

In conclusion, this article demonstrates that the rare apoE2(Arg145Cys) mutation is a common cause of type III HLP in our region and may be particularly prevalent in the black African population. No clear gene dose effect could be demonstrated between heterozygotes and homozygotes, and it appears that atherosclerosis is more likely in Westernized subjects. Family investigation revealed for the first time a clear dominant mode of inheritance with incomplete penetrance.

	Selected Abbreviations and Acronyms

 

apo = apolipoprotein BMI = body mass index -C = cholesterol HLP = hyperlipoproteinemia IHD = ischemic heart disease PAGE = polyacrylamide gel electrophoresis PCR = polymerase chain reaction RFLP = restriction fragment length polymorphism TC = total cholesterol TG = triglyceride

	Acknowledgments

 
The authors thank Pam Byrnes, Mary Moodie, Sheena Jones, and Felicity Leisegang for excellent technical assistance in this project.

Received May 17, 1996; accepted August 21, 1996.

	References

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