© 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 291
Ser 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).
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.
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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 291
Ser) 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%;
P
7.4x10-5) 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
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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.
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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.
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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
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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.
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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).
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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).
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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.
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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.
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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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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References |
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|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Mahley RW, Rall SC Jr. Type-III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Book Co; 1989:1195-1213.
2.
Weisgraber KH, Innerarity TL, Mahley RW.
Abnormal lipoprotein receptor-binding activity of the human
E apoprotein due to cysteine-arginine interchange at a single
site. J Biol Chem. 1982;257:2518-2521.
3.
Gregg RE, Zech LA, Schaefer EJ, Brewer HB Jr.
Type III hyperlipoproteinemia: defective
metabolism of an abnormal apolipoprotein E.
Science. 1981;211:584.
4. Utermann G, Vogelberg KH, Steinmetz A, Schoenborn W, Pruin N, Jaeschke M, Hees M, Canzler H. Polymorphism of apolipoprotein E, II: genetics of hyperlipoproteinemia type III. Clin Genet. 1979;15:37-62. [Medline] [Order article via Infotrieve]
5. Hazzard WR, Warnick GR, Utermann G, Albers JJ. Genetic transmission of isoapolipoprotein E phenotypes in a large kindred: relationship to dysbetalipoproteinemia and hyperlipidemia. Metabolism. 1981;30:79-88. [Medline] [Order article via Infotrieve]
6. Brunzell JD. Familial lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Book Co; 1989:1165-1180.
7.
Beisiegel U, Weber W, Bengtsson-Olivecrona G.
Lipoprotein lipase enhances the binding of chylomicrons to low
density lipoprotein receptor-related protein. Proc
Natl Acad Sci U S A. 1991;88:8342-8346.
8. Hayden MR, Ma Y. Molecular genetics of human lipoprotein lipase deficiency. Mol Cell Biochem. 1992;113:171-176. [Medline] [Order article via Infotrieve]
9. Ma Y, Ooi TC, Liu MS, Zhang HF, McPherson R, Edwards AL, Forsythe I, Frohlich J, Brunzell JD, Hayden MR. High frequency of mutations in the human lipoprotein lipase gene in pregnancy-induced chylomicronemia: possible association with apolipoprotein E2 isoform. J Lipid Res. 1994;35:1066-1075. [Abstract]
10. Hayden MR, Liu MS, Ma Y. Gene-environment interaction and plasma triglyceride levels: the crucial role of lipoprotein lipase. Clin Genet. 1994;46:15-18. [Medline] [Order article via Infotrieve]
11. Allain CC, Poon LS, Chan CS, Richmond W. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20:470-475. [Abstract]
12. Bucolo G, David H. Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem. 1973;19:476-482. [Abstract]
13.
Warnick GR, Albers JJ. Heparin-Mn2+
quantitation of high density lipoprotein cholesterol: an
ultrafiltration procedure for lipemic samples. Clin
Chem. 1978;24:900-904.
14. Havel RJ, Eder HA, Bradgon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
15. Main BF, Jones PJH, MacGillivray RTA, Banfield DK. Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers. J Lipid Res. 1991;32:183-187. [Abstract]
16. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI. J Lipid Res. 1990;31:545-548. [Abstract]
17. Müllenbach R, Lagoda PJL, Welter G. Technical tips: an efficient salt-chloroform extraction of DNA from blood and tissues. Trends Genet.1989;5:391.
18. Monsalve MV, Henderson HE, Roederer G, Julien P, Deeb S, Kastelein JJP, Peritz L, Devlin R, Bruin T, Ven Murthy MR, Gagne C, Davignon J, Lupien PJ, Brunzell JD, Hayden MR. A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries. J Clin Invest. 1990;86:728-734.
19.
Henderson HE, Ma Y, Hassan MF, Monsalve MV, Winkler F,
Gubernator K, Marais AD, Brunzell JD, Hayden MR. Amino acid
substitution (Ile194Thr) in exon 5 of the lipoprotein
lipase gene causes lipoprotein lipase deficiency in three unrelated
probands. J Clin Invest. 1991;87:2005-2011.
20. Seed B. An LFA-3 cDNA encodes a phospholipid-linked membrane protein homologous to its receptor CD2. Nature. 1987;329:840-842. [Medline] [Order article via Infotrieve]
21.
Ma Y, Bruin T, Tuzgol S, Wilson BI, Roederer G, Liu MS,
Davignon J, Kastelein JJP, Brunzell JD, Hayden MR. Two naturally
occurring mutations at the first and second bases of codon aspartic
acid 156 in the proposed catalytic triad of human lipoprotein lipase:
in vivo evidence that aspartic acid 156 is essential for
catalysis. J Biol Chem. 1992;267:1918-1923.
22.
Babirak SP, Iverius PH, Fujimoto WY, Brunzell JD.
Detection and characterization of heterozygous state for
lipoprotein lipase deficiency.
Arteriosclerosis. 1989;9:326-334.
23. Peterson J, Fujimoto WY, Brunzell JD. Human lipoprotein lipase: relationship of activity, heparin affinity, and conformation as studied with monoclonal antibodies. J Lipid Res. 1992;33:1165-1170. [Abstract]
24. Liu MS, Ma Y, Hayden MR, Brunzell JD. Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity. Biochim Biophys Acta. 1992;1128:113-115. [Medline] [Order article via Infotrieve]
25.
Iverius PH, Brunzell JD. Human adipose tissue
lipoprotein lipase: changes with feeding and relationship to post
heparin plasma enzyme. Am J Physiol. 1985;249:E107-E114.
26.
Smit M, de Knijff P, van der Kooij-Meijs E, Groenendijk
C, van den Maagdenberg AMJM, Gevers-Leuven JA, Stalenhoef AFH, Stuyt
PMJ, Frants RR, Havekes LM. Genetic
heterogeneity in familial dysbetalipoproteinemia: the
E2(Lys146Gln) variant results in dominant mode of
inheritance. J Lipid Res. 1990;31:45-53. [Abstract]
27.
Iverius PH, Ostlund-Lindqvist AM. Lipoprotein
lipase from bovine milk: isolation procedure, chemical
characterization, and molecular weight analysis.
J Biol Chem. 1976;251:7791-7795.
28. Osborne JC Jr, Bengtsson-Olivecrona G. Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation. Biochemistry. 1985;24:5606-5611. [Medline] [Order article via Infotrieve]
29. Reymer PWA, Gagne E, Groenemeyer BE, Zhang HF, Forsythe I, Jansen H, Seidell JC, Krombout D, Lie KE, Kastelein J, Hayden MR. A lipoprotein lipase mutation (Asn291Ser) is associated with reduced HDL cholesterol levels in premature atherosclerosis. Nat Genet. 1995;10:28-34. [Medline] [Order article via Infotrieve]
30.
Hata A, Ridinger DN, Sutherland S, Emi M, Zhang S,
Myers RL, Ren K, Cheng T, Inoue I, Wilson DE, Iverius PH, Lalouel JM.
Binding of lipoprotein lipase to heparin: identification of five
critical residues in two distinct segments of the amino-terminal
domain. J Biol Chem. 1993;268:8447-8457.
31. Ma Y, Henderson HE, Liu MS, Zhang HF, Forsythe IJ, Clarke-Lewis I, Hayden MR, Brunzell JD. Mutagenesis in four candidate heparin binding regions (residues 279-282, 291-304, 390-393, and 439-448) and identification of residues affecting heparin binding of human lipoprotein lipase. J Lipid Res. 1994;35:2049-2059. [Abstract]
32. Tybjerg-Hansen A, Gallagher J, Vincent J, Houlston R, Talmud P, Dunning AM, Seed M, Hamsten A, Humphries SE, Myant NB. Familial defective apolipoprotein B-100: detection in the United Kingdom and Scandinavia, and clinical characteristics of ten cases. Atherosclerosis. 1990;80:235-242. [Medline] [Order article via Infotrieve]
33.
Feussner G, Schuster H. Screening for the
apolipoprotein B-100 arginine3500glutamine mutation in patients with
type III hyperlipoproteinemia.
Clin Genet. 1992;42:302-305. [Medline]
[Order article via Infotrieve]
34.
Zhao SP, Smelt AHM, Leuven JAG, van den Maagdenberg
AMJM, van der Laarse A, van't Hooft FM. Lipoproteins in
familial dysbetalipoproteinemia: variation of serum
cholesterol level associated with VLDL
concentration. Arterioscler Thromb. 1993;13:316-323.
35. Minnich A, Kessling A, Roy M, Giry C, DeLangavant G, Lavigne J, Lussier-Cacan S, Davignon J. Prevalence of alleles encoding defective lipoprotein lipase in hypertriglyceridemic patients of French Canadian descent. J Lipid Res. 1995;36:117-124. [Abstract]
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