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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1695-1703.)
© 1995 American Heart Association, Inc.

Articles

Patients With ApoE3 Deficiency (E2/2, E3/2, and E4/2) Who Manifest With Hyperlipidemia Have Increased Frequency of an Asn 291Ser Mutation in the Human LPL Gene

Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 8-11, 1993, and published in abstract form (Circulation. 1993;88[suppl I]:I-179).

Hanfang Zhang; Paul W. A. Reymer; Ming-Sun Liu; Ian J. Forsythe; Bjorn E. Groenemeyer; Jiri Frohlich; John D. Brunzell; John J. P. Kastelein; Michael R. Hayden; Yuanhong Ma

From the Department of Medicine (H.Z., Y.M.), the Department of Medical Genetics (H.Z., M.-S.L., I.J.F., M.R.H), and the Academic Medical Center, Amsterdam, The Netherlands (P.W.A.R, B.E.G, J.J.P.K.); the Department of Pathology (J.F.), University of British Columbia, Vancouver, Canada; and the Department of Medicine, University of Washington, Seattle (J.D.B.).

Correspondence to Dr Yuanhong Ma, Department of Medicine, Rm 416-2125 East Mall, NCE Bldg, Vancouver, BC V6T 1Z4, Canada.

	Abstract

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Abstract Approximately 1% to 2% of persons in the general population are homozygous for a lipoprotein receptor–binding defective form of apoE (apoE2/2). However, only a small percentage (2% to 5%) of all apoE2/2 homozygotes develop type III hyperlipoproteinemia. Interaction with other genetic and environmental factors are required for the expression of this lipid abnormality. We sought to investigate the possible role of LPL gene mutations in the development of hyperlipoproteinemia in apoE2/2 homozygotes and in apoE2 heterozygotes. As a first step, we performed DNA sequence analysis of all 10 LPL coding exons in 2 patients with the apoE2/2 genotype who had type III hyperlipoproteinemia and identified a single missense mutation (Asn 291Ser) in exon 6 of the LPL gene. The mutation was then found in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype (allele frequency=13.9%; P7.4x10^-5) and 6 of 22 hyperlipidemic E2 heterozygous patients with the apoE3/2 and E4/2 genotype (allele frequency=13.6%; P=2.2x10^-5). In contrast, this mutation was found in only 3 of 230 normolipidemic controls (allele frequency=0.7%). In vitro mutagenesis studies revealed that the Asn 291Ser mutant LPL had approximately 60% of LPL catalytic activity and approximately 70% of specific activity compared with wild-type LPL. The heparin-binding affinity of the mutant LPL was not impaired. Our data suggest that the Asn 291Ser substitution is likely to be a significant predisposing factor contributing to the expression of different forms of hyperlipidemia when associated with other genetic factors such as the presence of apoE2.

Key Words: hyperlipoproteinemia • lipoprotein lipase • missense mutations • apoE • gene-gene interaction

	Introduction

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ApoE is a 299–amino acid glycoprotein that is a constituent of chylomicrons, VLDLs, IDLs, and HDLs and is a ligand for the receptor-mediated hepatic uptake of chylomicron and VLDL remnant particles.¹ ApoE is genetically polymorphic, and the most common allele, apoE3 (Cys 112, Arg 158), has a frequency of approximately 75% in the general population, whereas the other two less common alleles, apoE4 (Arg 112, Arg 158) and apoE2 (Cys 112, Cys 158), have frequencies of approximately 15% and 10%, respectively.¹

The apoE2 isoform has been shown both in vitro and in vivo to be defective in mediating the clearance of remnant lipoproteins by hepatic receptors.^{2 3} Some apoE2/2 homozygotes develop type III hyperlipoproteinemia, which is a distinctive genetic disorder of lipoprotein metabolism characterized by the clinically significant accumulation of remnant lipoproteins due to defective hepatic removal of these lipoproteins.¹ These patients have both increased cholesterol and TG levels. The remnant lipoprotein, also known as ß-VLDL because of its ß-electrophoretic mobility, is the major abnormal lipoprotein in type III hyperlipoproteinemia. In addition, the ß-VLDL is enriched in cholesterol, as reflected by a ratio of VLDL cholesterol to plasma TG of more than 0.3. Patients with type III hyperlipoproteinemia are predisposed to the development of premature atherosclerosis.

Approximately 1% of individuals in the general population are apoE2/2 homozygotes. However, only a small percentage (2% to 5%) of these apoE2/2 homozygotes develop type III hyperlipoproteinemia. Utermann et al⁴ and Hazzard et al⁵ suggested that homozygosity for apoE2 is a necessary but insufficient genetic influence and that additional genetic or environmental factors are required for the expression of type III hyperlipoproteinemia in apo E2/2 homozygotes. Until now, no other abnormalities in other genes have been identified.

LPL plays a crucial role in the metabolism of chylomicrons and VLDL as the rate-limiting enzyme in the hydrolysis of the TG core in these lipoproteins.⁶ Another role for LPL in lipid metabolism has recently been postulated: namely, that LPL functions as a ligand for LRP to mediate the uptake of chylomicron and VLDL remnants.⁷ We therefore hypothesized that genetic defects in the LPL gene might be one of the genetic factors contributing to the expression of type III hyperlipoproteinemia in apoE2/2 homozygotes.

Genetic defects in LPL have been studied extensively in patients with complete deficiency of LPL (type I chylomicronemia) in recent years (for review see Reference 8⁸ ). Patients with complete LPL deficiency often have no, or extremely low, LPL activity and fasting plasma TG level usually above 1500 mg/dL (17 mmol/L), which is frequently associated with recurrent episodes of abdominal pain, pancreatitis, and eruptive xanthomas from early childhood.⁶ These patients have been shown to be either homozygous for a single mutation or compound heterozygous for different mutations in the LPL gene. More than 40 mutations in the LPL gene have been reported in patients with LPL deficiency, and in most cases these mutations were found to cause a completely catalytically defective LPL protein.⁸

Although complete LPL deficiency is rare, partial LPL deficiency due to heterozygosity for a mutation causing defective LPL is more frequent and may sometimes be associated with mild hypertriglyceridemia, especially when other genetic or environmental factors are also present in the same patient.^{6 9 10} We have recently reported that partial LPL deficiency in apoE2 heterozygotes is likely to be a common factor in pregnancy-induced severe hypertriglyceridemia.⁹

Because of the crucial role played by LPL in chylomicron and VLDL metabolism, the relatively high frequency of heterozygosity for LPL mutations in the general population, and the possible synergistic interaction between LPL and apoE, we hypothesized that LPL mutations in the heterozygote state might be one of the additional genetic factors required for the expression of type III hyperlipoproteinemia in some apoE2/2 homozygotes. In this article, we report the analysis of the LPL gene in hyperlipidemic patients with the apoE2/2, E3/2, or E4/2 genotype. We found significantly increased frequency of an Asn 291Ser mutation in the LPL gene in these patients. Our study suggests that the Asn 291Ser mutation is likely to be a significant predisposing factor for the expression of type III hyperlipoproteinemia in some apoE2/2 homozygotes and for the expression of hyperlipidemia in apoE2 heterozygotes.

	Methods

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Patient Selection
Sixty Caucasian patients (39 male, 21 female) were consecutively selected initially as patients with type III hyperlipoproteinemia. These included 15 patients of mixed Caucasian origin from the University Hospital Lipid Clinic in Vancouver, Canada, and 45 patients of Dutch origin from the Academic Medical Center in Amsterdam, The Netherlands. The mean age of these patients was (mean±SD) 50±12 years and ranged from 14 to 69 years. The initial diagnosis of type III hyperlipoproteinemia was based on the observations that (1) most of the patients had both hypercholesterolemia and hypertriglyceridemia and (2) all were found, by use of standard isoelectric focusing gel analysis, to have the apoE2/2 phenotype. However, subsequent apoE genotyping with two different genotyping methods revealed that 38 had the apoE2/2 genotype, 17 had the apoE3/2 genotype, and 5 had the apoE4/2 genotype. For the clarity of this report, we classified these patients into three different groups on the basis of their apoE genotypes (Table 1). The type III group consisted of 18 patients with hyperlipidemia, E2/2 genotype, and a VLDL-C/TG ratio of >0.3; the HLP-E2/2 group comprised 20 patients with hyperlipidemia and apoE2/2 genotype but either with a VLDL-C/TG ratio of <0.3 or without the ratio data; the HLP-E2 group contained 22 hyperlipidemic E2 heterozygotes with apoE3/2 or E4/2 genotypes. Detailed clinical and biochemical data on all patients are provided in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical and Biochemical Characteristics of 60 Patients

Control Group
The control group (230 normolipidemic persons between the ages of 23 and 60 years [mean±SD, 45±9 years]) included 80 randomly selected individuals of Western European origin from Vancouver and 150 randomly selected persons of Dutch origin from Amsterdam. They had concentrations of plasma TG of less than 205 mg/dL (2.3 mmol/L), of HDL cholesterol more than 37 mg/dL (0.95 mmol/L), and of LDL cholesterol less than 190 mg/dL (5 mmol/L). In addition, they met the following criteria: (1) a history of good health and (2) abstinence from drugs known to affect serum lipids.

Lipid and Lipoprotein Measurements
Blood samples were collected from the patients and control subjects after a 12- to 16-hour overnight fast. Fasting plasma TG, HDL cholesterol, and LDL cholesterol levels were measured as previously described.^{11 12 13} For some patients VLDL cholesterol and LDL cholesterol were also determined by sequential ultracentrifugation as described.¹⁴

ApoE Genotyping
ApoE genotyping was initially performed with a mismatch PCR method as previously described by Main et al.¹⁵ All apoE genotyping was confirmed by another method with PCR followed by cleavage with Hha I restriction enzyme as described.¹⁶ Both methods can be used to detect the amino acid composition at residues 112 and 158 of the apoE gene.

DNA Analysis
Genomic DNA was isolated from white blood cells of the patients as previously described.¹⁷ Each of the 10 exons of LPL was individually amplified from 0.5 to 1 µg of genomic DNA by use of PCR as previously described.^{18 19} The amplified exons were then purified and sequenced either directly or after cloning into a TA cloning vector (Invitrogen Inc).

Mutation Detection With Mismatch PCR Followed by RsaI Restriction Digestion
Exon 6 of the LPL gene from all 60 patients and 230 normolipidemic control subjects was amplified with a 5'-PCR primer located in intron 5 near the 5' boundary of exon 6 (5'-GCCGAGATACAATCTTGGTG-3') and a 3'-mismatch PCR primer located in exon 6 near the Asn 291Ser mutation (5'-CTGCTTCTTTTGGCTCTGACTGTA-3'). PCR amplification reactions were performed with 0.5 µg of genomic DNA in PCR buffer containing 1.5 mmol/L MgCl₂, 200 µmol/L dNTPs, 1 µmol/L of each primer, and 2.5 U Taq polymerase (BRL). The reaction mixture was denatured at 95°C for 1 minute, annealed at 51°C for 1 minute, and extended at 72°C for 45 seconds for a total of 35 cycles. The PCR product (20 µL) was then digested with 10 U Rsa I enzyme, 3.5 µL 10x reaction buffer 1 (BRL), and 9.5 µL water at 37°C for 2 hours. The digested fragments were then separated on 2% agarose gels.

In Vitro Site-Directed Mutagenesis and Expression in COS Cells
A 1.6-kb cDNA fragment containing the entire coding sequence of the LPL gene was cloned into a dual function vector (CDM8) for both mutagenesis and expression.²⁰ The sequences of the mutagenesis oligonucleotide for Asn 291Ser mutation was 5'-CTATGAGATCAGTAAAGTCAGA-3'. Mutagenesis was performed as previously described.²¹ Mutant clones were identified by oligonucleotide hybridization and confirmed by DNA sequencing. COS cell tranfections with purified phagemid DNA carrying either the mutant or wtLPL cDNA were performed either by electroporation as previously described²¹ or by use of a lipofectin method (GIBCO-BRL). In brief, 10 µg of DNA was used for electroporation and the transfected cells were plated in 15-cm culture dishes. For the lipofectin method, 1.5 µg of DNA was used in each of the six-well culture plates (Falcon) according to the manufacturer's instructions (GIBCO-BRL).

Measurement of LPL Mass and Catalytic Activity
LPL mass levels in COS medium were measured by an ELISA method with two different monoclonal antibodies, 5F9 and 5D2, as previously described.^{22 23} In brief, the ELISA plate (F16 MAXISORP, Nunc) was coated with 5F9 as capturing antibody and another monoclonal antibody, 5D2 conjugated with HRP, was used to detect the bound LPL. We have previously reported that the 5D2 antibody recognizes an epitope located between residues 396 and 405 of human LPL,²⁴ but the epitope for the 5F9 antibody remains unknown. The 5F9 antibody only recognizes partially denatured LPLs, such as dissociated LPL monomers, and was therefore used to determine the amount of LPL monomer in the COS medium. Specifically, the microtiter plate was coated with 5F9 and incubated for 4 hours at 37°C. Purified bovine LPL (used as standard controls) or COS medium samples were added to each well and incubated for 18 hours at 4°C. The wells were then washed to remove the unbound LPL and the detecting antibody, 5D2 conjugated with HRP, was added as previously described.^{22 23} After a 4-hour incubation at room temperature, the wells were washed five times and substrate was added for color development. To determine the amount of LPL dimer, an aliquot of the COS medium was treated with 1 mol/L guanidine hydrochloride to allow dissociation of the dimer into monomer, and the total amount of the monomer in the sample was then determined.²³ The difference between samples with and without the guanadine hydrochloride treatment represents the amount of LPL dimer in the COS medium. LPL lipolytic activities were measured by use of a radiolabeled tri-1-[¹⁴C]oleate phospholipid emulsion as previously described.²⁵

Heparin-Sepharose Chromatography
Heparin-binding affinity of the in vitro–expressed wtLPL and Asn 291Ser mutant LPL was assessed on a Bio-Rad Econo-Column packed with 1 mL of heparin-Sepharose CL-6B (Pharmacia) as previously described.²³ Aliquots of COS cell medium containing 300 to 400 ng of LPL dimer mass were applied to the column at a flow rate of 0.25 mL/min and eluted, without an intermediate washing step, with a linear NaCl gradient from 0.4 mol/L to 1.8 mol/L. Twenty-six fractions of 1.5 mL each were collected, and LPL mass and activity and eluent conductivity were determined for each fraction as described.²³

	Results

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Biochemical Measurements
The results of fasting plasma TG, total cholesterol, and HDL cholesterol levels for all 60 patients are listed in Table 1. VLDL cholesterol levels were determined after ultracentrifugation (Table 1). Almost all patients had increased levels of plasma TG and most of them also had increased cholesterol levels. Eighteen apo E2/2 homozygotes also had ratios of VLDL-C/total TG of more than 0.3, suggesting the presence of ß-VLDL. In addition, some patients with the apoE2/2 genotype also had other factors such as obesity (BMI >26), hyperuricemia, hypothyroidism, or diabetes, which are often seen in patients with type III hyperlipoproteinemia.

ApoE Genotyping
All 60 patients were selected based on their apoE2/2 phenotype. However, 17 were found to be apoE3/2 and 5 were apoE4/2 by genotype analysis with two different methods. One of the patients with E3/2 has previously been shown to carry a Lys 146Gln substitution on the apoE3* allele, which results in the loss of a positive charge in the apoE3 (Lys 146Gln) protein (apoE3*).²⁶ Thus, the apoE3* protein has the same number of charge residues as the apoE2 protein and is indistinguishable from the apoE2 protein on isoelectric focusing gel used for apoE phenotyping. No other patient in this study was found to carry the Lys 146Gln mutation (data not shown). Among the 38 patients with the apoE2/2 genotype, 18 who had a VLDL-C/total TG ratio of more than 0.3 were diagnosed as having type III hyperlipoproteinemia, and the remaining 20 patients who either had a VLDL-C/total TG ratio of more than 0.3 or did not have the ratio data were classified as hyperlipidemic apo E2/2 homozygotes. Twenty-two patients with the apoE3/2 or E4/2 genotype were classified as hyperlipidemic E2 heterozygotes (Table 1).

DNA Analysis of the LPL Gene
Because the vast majority of the mutations in the LPL gene that have previously been described are clustered in exons 4, 5, and 6, we initially performed DNA sequence analysis of exons 4, 5, and 6 of 6 patients with type III hyperlipoproteinemia. An A to G transition at the second base of codon 291 in exon 6 resulting in an asparagine (AAT) to serine (AGT) substitution was identified in 2 of the 6 patients (Fig 1). DNA sequence analysis of all 10 exons and intron-exon boundaries in the LPL gene was subsequently performed for these 2 patients and no other DNA alteration was identified. Several independent PCR amplifications of exon 6 and subsequent DNA sequencing of both the coding and noncoding strands were performed to confirm the presence of the Asn 291Ser mutation in these 2 type III patients.

View larger version (25K): [in this window] [in a new window]   Figure 1. Photograph shows DNA sequence of exon 6 spanning residue Asn 291 of the LPL gene. Exon 6 DNA from a type III hyperlipidemia patient and from a normal control subject was PCR amplified individually and sequenced after cloning into a TA vector. Plasmid DNA was individually purified from 10 positive clones for each exon and sequenced for both coding and noncoding strands. An AG transition in the coding strand (TC in the noncoding strand) resulting in a substitution of serine for asparagine at residue 291 is highlighted in the boxes. The 10 clones sequenced were found to carry either the Asn 291Ser mutant gene (4 of the 10) or the normal LPL gene (6 of the 10), indicating that this patient is a heterozygote for the Asn 291Ser mutation.

Detection of the Asn 291Ser Mutation by Mismatch PCR and Rsa I Digestion
The finding that 2 of the 6 randomly selected type III patients both carried the Asn 291Ser mutation suggested that this mutation might be a relatively common one in this group of patients. To detect this mutation in a larger group of patients and control subjects, we designed a mismatch PCR primer as the 3'-PCR primer that was used together with the normal 5'-PCR primer for the amplification of a 238-bp fragment in exon 6 of the LPL gene. The A to G mutation resulting in the Asn 291Ser substitution is located at nucleotide 1127 of the LPL gene. The use of a mismatch primer generates a C instead of the normal A (the mismatch) at nucleotide 1130 in the PCR fragments amplified from both the mutant and normal alleles. Thus, the PCR fragment from the mutant allele will have a 5'-GTAC-3' sequence in this region between nucleotides 1127 and 1130 (G₁₁₂₇ from the N291S mutation and C₁₁₃₀ from the mismatch) and can be cleaved into two fragments of 215 bp and 23 bp by the Rsa I digestion (Fig 2). However, the PCR fragment from the normal allele will have a 5'-ATAC-3' sequence in this region (A₁₁₂₇ from the normal allele and C₁₁₃₀ from the mismatch) and cannot be cleaved by the Rsa I digestion, therefore remaining as a single 238-bp fragment (Fig 2). Family members of 4 of the patients who are heterozygous for the Asn 291Ser mutation were also analyzed to confirm that the screening method meets the criteria of mendelian inheritance (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 2. Photograph shows detection of the Asn 291Ser mutation by PCR and Rsa I restriction digestion. Exon 6 DNA was amplified using the normal 5'-PCR primer and a 3'-mismatch PCR primer near residue 291 of the LPL gene. The 238-bp PCR product from the normal allele remains intact after the Rsa I digestion, whereas the same PCR product from the Asn 291Ser mutant allele is cut into a 215-bp fragment and a 23-bp fragment. Lanes 1, 2, 5, 6, and 7 are the digested PCR DNA from patients who do not have the Asn 291Ser mutation; lanes 3, 4, and 8 are the digested PCR DNA from Asn 291Ser heterozygotes.

The results of the Asn 291Ser screening in the patient and control groups are summarized in Table 2. Among 18 type III patients with the apoE2/2 or apoE3*/2 genotype, 5 (allele frequency=13.9%) were found to be heterozygotes for the Asn 291Ser mutation. In 22 hyperlipidemic E2 heterozygotes with either the apoE3/2 or E4/2 genotype, 6 (allele frequency=13.6%) were heterozygotes for this mutation. However, this mutation was not found in 20 hyperlipidemic apoE2/2 homozygotes who do not have classical type III hyperlipoproteinemia (ie, those who did not have a VLDL-C/TG ratio of more than 0.3). In the normolipidemic control group, 3 heterozygotes were found among 230 normolipidemic control subjects (allele frequency=0.7%). Of the 3 Asn 291Ser heterozygotes from the control group, 2 had the apoE3/3 genotype and 1 had the apoE4/3 genotype. No homozygote for the Asn 291Ser mutation was identified among either the patients or the controls. The frequency of the Asn 291Ser mutation is significantly higher in both the type III patients (P7.4x10^-5) and the hyperlipidemic E2 heterozygotes (P2.2x10^-5) than in the control group (Table 2).

View this table: [in this window] [in a new window]   Table 2. Allele Frequency of the Asn 291Ser Mutation in Patients and Control Subjects

In Vitro Site-Directed Mutagenesis and Expression in COS Cells
To determine whether the Asn 291Ser substitution altered LPL mass and activity level in vitro, we performed in vitro site-directed mutagenesis studies to reproduce the Asn 291Ser mutation. Phagemid DNA from the Asn 291Ser mutant and from a wtLPL cDNA clone was purified and used to transfect COS-1 cells. Functional LPL is in a dimer form.^{23 27 28} LPL total, dimer, and monomer mass and LPL activity in the transfected COS cell medium were assayed for each set of wtLPL and mutant cDNA. In four sets of dishes from two separate transfection experiments, the total Asn 291Ser mutant LPL mass levels were similar to the normal wtLPL levels (Table 3), which suggests that the Asn 291Ser mutant LPL can be effectively secreted into the medium. However, the monomer Asn 291Ser LPL mass was significantly increased, whereas the ratio of active dimer versus inactive monomer was significantly decreased. This suggests that the Asn 291Ser mutant LPL may be less stable and have a higher tendency to dissociate from dimers to monomers.

View this table: [in this window] [in a new window]   Table 3. LPL Mass and Activity in COS Medium in Transfection Experiments With Single Constructs

In the four sets of parallel transfections, the LPL activity levels for the Asn 291Ser mutant versus its wtLPL control were 66.0 versus 93.0 nmol · min^-1 · mL^-1 (71%), 43.7 versus 80.0 nmol · min^-1 · mL^-1 (55%), 34.4 versus 79.5 nmol · min^-1 · mL^-1 (43%), and 61.4 versus 90.5 nmol · min^-1 · mL^-1 (68%). The mean activity for the mutant LPL is 60% of the wtLPL activity level, and this is statistically significant (Table 3). The specific activity levels of the mutant LPL versus wtLPL were 0.22 versus 0.28 nmol FFA · min^-1 · ng^-1 (78%), 0.19 versus 0.3 nmol FFA · min^-1 · ng^-1 (63%), 0.11 versus 0.25 nmol FFA · min^-1 · ng^-1 (44%), and 0.22 versus 0.28 nmol FFA · min^-1 · ng^-1 (78%). This reduction in specific activity is also consistent and statistically significant (Table 3). The observation that the mutant LPL activity and specific activity were consistently lower in each set of transfections suggests that both the LPL activity and specific activity for the Asn 291Ser mutant are very likely to be reduced, but the exact percentage of the reduction cannot be certain because of the fairly large variations. The observed reduction in vitro is consistent with two related in vivo studies that showed that patients with the Asn 291Ser mutation had significantly reduced LPL activity in their postheparin plasma.^{9 29}

Heparin-Sepharose Chromatography
One of the two heparin-binding sites has been located between residues 292 and 304 of the LPL protein.^{30 31} To examine whether the Asn 291Ser mutation has an effect on the heparin-binding affinity of the mutant LPL, we performed heparin-Sepharose chromatography for both wtLPL and Asn 291Ser mutant LPL expressed in COS medium. Both wtLPL dimer peak and Asn 291Ser dimer peaks eluted from the column in similar fractions, indicating that the Asn 291Ser mutation does not significantly alter the heparin-binding affinity of LPL. Similarly, the inactive monomer peak from both the wtLPL and Asn 291Ser mutant LPL eluted from the column in similar fractions.

	Discussion

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Although homozygosity for apoE2 is a necessary genetic factor, additional genetic or environmental factors are required for the expression of type III hyperlipoproteinemia because only 2% to 5% of apoE2/2 homozygotes develop this type of hyperlipoproteinemia.¹ Further support for other genetic factors predisposing to type III hyperlipoproteinemia comes from studies demonstrating that family members of apoE2/2 patients with type III hyperlipoproteinemia have relatives with other forms of hyperlipidemia such as hypertriglyceridemia, hypercholesterolemia, or familial combined hyperlipoproteinemia, whereas apoE2/2 homozygotes without type III hyperlipoproteinemia do not have relatives with lipid abnormalities.^{4 5} The coexistence of these other genetic factors and the apoE2/2 isoform leads to type III hyperlipoproteinemia in the probands, whereas the cosegregation of the same genetic factors with other apoE isoforms may result in other lipoprotein phenotypes in relatives.

The molecular basis of these other genetic factors has not been identified. A mutation in the apoB gene, Arg 3500Glu, which leads to defective binding ability to the LDL receptor, has previously been suggested as a possible secondary genetic factor in type III hyperlipoproteinemia. However, this mutation was not found in 30 patients with type III hyperlipoproteinemia who had the apoE2/2 phenotype.³² Another study of patients of German origin also failed to detect the Arg 3500Glu mutation in 43 patients with type III hyperlipoproteinemia.³³ These results suggest that this mutation in apoB is not a common predisposing genetic factor leading to type III hyperlipoproteinemia.

In this article, we report the identification of an Asn 291Ser mutation in the LPL gene in 5 of 18 patients with type III hyperlipoproteinemia who had the apoE2/2 genotype and in 6 of 22 hyperlipidemic E2 heterozygotes who had the apoE3/2 or E4/2 genotypes. In contrast, this Asn 291Ser mutation was found in 3 of the 230 normolipidemic control subjects. This suggests that the Asn 291Ser substitution in the LPL gene is unlikely to be a common DNA polymorphism of the LPL gene in this population but that the Asn 291Ser mutation that leads to functional defect is likely to be a secondary genetic factor contributing to the expression of type III hyperlipoproteinemia when present in apoE2/2 homozygotes. However, the same mutation, when present in apoE2 heterozygotes, may make individuals more susceptible to other forms of hyperlipidemia.

Among the 38 apoE2/2 patients included in this study, 18 had classic type III hyperlipoproteinemia with the accumulation of ß-VLDL as indicated by a VLDL-C/total TG ratio of more than 0.3. Five of these 18 patients carried the Asn 291Ser mutation. Interestingly, the same mutation was not found in the remaining 20 hyperlipidemic apoE2/2 homozygotes who do not appear to have significant accumulation of ß-VLDL (VLDL-C/TG <0.3). The observation that the frequency of the Asn 291Ser mutation is not increased in this group might suggest that apoE2/2 homozygotes who do not have the Asn 291Ser mutation might have a lower risk of developing classic type III hyperlipoproteinemia. Clearly, additional studies including more apoE2/2 homozygotes are required to examine this hypothesis.

Although none of the 11 Asn 291Ser heterozygotes in this study were available for postheparin LPL mass and activity measurements, in two related studies we have clearly shown that Asn 291Ser heterozygotes have significantly reduced postheparin plasma LPL activity compared with control subjects.^{9 29} To determine whether the Asn 291Ser mutation affects LPL mass and activity in vitro, we performed site-directed mutagenesis and expression experiments. In transfection experiments with Asn 291Ser mutant cDNA, we consistently found significantly reduced LPL activity (about 60% of normal) and reduced specific activity (about 70% of normal) (Table 3). These findings are consistent with the reduced levels of postheparin LPL activity seen in Asn 291Ser patients.

The Asn 291Ser mutation is likely to be the cause for the reduced LPL activity and specific activity seen in the patients. This is supported by the fact that (1) the Asn 291 residue is conserved in all LPL genes from different species, including mouse, cattle, guinea pig, chicken and cat; (2) consistently reduced LPL activity and specific activity were observed in the in vitro transfection experiments; and (3) we have sequenced the entire coding region of the LPL gene and did not find any other mutation. However, a specific mutation in the noncoding region of the LPL gene that is in linkage disequilibrium with the Asn 291Ser mutation has not been excluded.

In vitro, heparin binding is a necessary step for LPL-mediated binding of remnant particles to LRP.⁷ One of the heparin-binding domains of LPL has recently been located in a segment between amino acids 292 and 304.^{30 31} In this study, both Asn 291Ser mutant monomer and dimer showed normal heparin-binding affinity compared with the wtLPL. In addition, the Asn 291Ser mutant LPL appears to be normal in mediating the binding of ß-VLDL to HepG2 cells in vitro (A. Krapp, PhD, et al, unpublished data, 1995). Thus, the possible involvement of Asn 291Ser mutant LPL in the pathogenesis of type III is probably not due to further reduction of ß-VLDL clearance in apoE2/2 homozygotes.

The exact mechanism by which the Asn 291Ser mutation in the LPL gene contributes to the development of type III hyperlipoproteinemia remains to be elucidated. The normal heparin-binding and LRP-binding affinity of the Asn 291Ser mutant LPL suggests that this mutation does not result in defective clearance of remnants through LRP. Also, because completely defective lipolysis is expected to cause accumulation of large TG-rich chylomicrons and VLDL and therefore reduced levels of smaller cholesterol-rich remnants, it is not clear how the partially reduced LPL activity seen in Asn 291Ser heterozygotes contributes to the accumulation of cholesterol-rich remnant particles seen in patients with type III hyperlipoproteinemia. One possibility is that the combined defects of lipolysis due to the Asn 291Ser mutation and reduced uptake of E2-containing remnants by the liver due to homozygosity for apoE2/2 might have a feedback effect on the production rate and/or the lipid composition of VLDL. Zhao et al³⁴ recently reported that many patients with type III hyperlipoproteinemia had markedly increased levels (more than 10-fold) of large cholesterol-rich VLDL compared with normolipidemic control subjects. The exact mechanism for the overproduction of these cholesterol-rich VLDL remains unknown. Finally, because the LPL activity assays in our study were performed on artificial lipid substrates that do not contain apolipoproteins such as apoE that are present in native chylomicron and VLDL substrates, we still do not know whether the Asn 291Ser mutant LPL is also defective in either the binding or hydrolysis of apoE2-containing chylomicrons and VLDL. Very little is known about LPL function on lipoproteins containing different apoE isoforms, and further studies will be required to test this hypothesis.

Our genetic study indicates that the Asn 291Ser mutation in the LPL gene was probably a predisposing factor for the development of type III hyperlipoproteinemia in 5 of the 18 apoE2/2 homozygotes and for the expression of hyperlipidemia in 6 of the 22 apoE3/2 and E4/2 heterozygotes in this study. The finding that 3 of 230 normolipidemic control subjects also carried the Asn 291Ser mutation suggests that the presence of that mutation alone may not be sufficient for the development of hyperlipidemia. Other genetic factors, such as the apoE2 genotype, or environmental factors may also be required for the expression of hyperlipidemia. This hypothesis is further supported by our previous study, which showed that the Asn 291Ser mutation and the apoE3/2 genotype were found in a patient with pregnancy-induced hypertriglyceridemia.⁹ In another study, the Asn 291Ser mutation was also found in a patient with alcohol-induced hypertriglyceridemia (Y. Ma, PhD, et al, unpublished data, 1995). Very recently, the Asn 291Ser mutation was found in 8 of the 95 French-Canadian patients with type IV hyperlipoproteinemia but not in any of the 72 normolipidemic French-Canadian control subjects.³⁵ Because of the potential gene-gene and gene-environment interaction involving the Asn 291Ser mutation in the LPL gene, it is anticipated that this mutation may be found in several different forms of hyperlipoproteinemia.

	Selected Abbreviations and Acronyms

 

COS = SV40 transformed African Green Monkey kidney ELISA = enzyme-linked immunosorbent assay HLP-E2 group = group of hyperlipidemic E2 heterozygotes HLP-E2/2 group = group of hyperlipidemic apoE2/2 homozygotes LPL = lipoprotein lipase LRP = LDL receptor–related protein PCR = polymerase chain reaction TG = triglycerides type III group = group of patients with type III hyperlipoproteinemia wtLPL = wild-type LPL

	Acknowledgments

 
This work is supported by grants from the Medical Research Council of Canada and the British Columbia Heart and Stroke Foundation (to Y.M. and M.R.H.); The Canadian Genetic Disease Network (to M.R.H.); and the National Institutes of Health (DK-02456) (to J.D.B.). Dr Michael Hayden is an established investigator of the British Columbia Children's Hospital. Dr Yuanhong Ma is an investigator supported by a scholarship from the Medical Research Council of Canada. We thank Steve Hashimoto and Alegria Albers for excellent technical assistance.

Received July 25, 1994; accepted July 10, 1995.

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