Clinical Chemistry 47: 438-443, 2001;
(Clinical Chemistry. 2001;47:438-443.)
© 2001 American Association for Clinical Chemistry, Inc.
Compound Heterozygous Familial Hypercholesterolemia and Familial Defective Apolipoprotein B-100 Produce Exaggerated Hypercholesterolemia
E. Shyong Tai1,2,a,
Evelyn S.C. Koay2,3,2,
Edmund Chan1,
Tzer Jing Seng3,
Lih Ming Loh1,
Sunil K. Sethi2,3 and
Chee Eng Tan1
1
Lipid Unit, Department of Endocrinology, Singapore General Hospital, Singapore 169608, Republic of Singapore.
2
Department of Laboratory Medicine, National University
Hospital, Singapore 119074, Republic of Singapore.
3
Department of Pathology, National University of
Singapore, Singapore 119260, Republic of Singapore.
a Author for correspondence. Fax 65-227-3576; e-mail
eshyong{at}pacific.net.sg.
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Abstract
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Background: Familial hypercholesterolemia (FH) and familial
defective apolipoprotein B-100 (FDB) represent ligand-receptor
disorders that are complementary. Individuals with both FH and FDB are
unusual. We report a family with both disorders and the impact of the
mutations on the phenotypes of the family members.
Methods: We used single strand conformation polymorphism (SSCP)
and denaturing gradient gel electrophoresis (DGGE) for genetic analysis
of all 18 exons and the promoter region of the LDL receptor and
DGGE for genetic analysis of the apolipoprotein B-100 (apo B-100) gene.
The functional significance of the apo B-100 mutation was studied using
a U937 cell proliferation assay. Fasting serum lipid profiles were
determined for the index case and seven first-degree relatives.
Results: One of the patient’s sisters had a missense mutation
(Asp407
Lys) in exon 9 of the LDL receptor and a serum
LDL-cholesterol concentration of 4.07 mmol/L. Four other first-degree
relatives had hyperlipidemia but no LDL-receptor mutation.
However, these subjects had a mutation of the apo B-100 gene
(Arg3500Trp). The cell proliferation rate of U937 cells
fed with LDL from other subjects with the same mutation was fourfold
less than that of controls. The index case had both FH- and FDB-related
mutations. Her serum LDL-cholesterol (9.47 mmol/L) was higher than all
other relatives tested.
Conclusions: Existence of both FH and FDB should be considered in
families with LDL-receptor mutations in some but not all individuals
with hypercholesterolemia or when some individuals in families with FH
exhibit exaggerated hypercholesterolemia.
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Introduction
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Familial hypercholesterolemia
(FH),1
an autosomally dominant inherited defect of LDL receptors,
produces premature coronary artery disease (CAD) and occurs in 1 in 500
individuals (1)(2). Familial defective
apolipoprotein B-100 (FDB) results from a mutation at the
apolipoprotein B-100 (apo B-100) locus on chromosome 2, leading to
defective binding of LDL to the LDL receptor (3). It also
occurs in 
1 in 500 individuals and leads to premature CAD. FDB
cannot be differentiated from FH phenotypically (4).
Subjects with both FH and FDB are rare, and there have been only three
reports in the literature of such compound heterozygotes
(5)(6)(7).
Here we describe a proband who was identified to have both FH and FDB,
detected as part of a family screening program for the detection of FH.
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Materials and Methods
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The proband was identified based on the presence of severe
hypercholesterolemia and the presence of tendon xanthomata in
accordance with established diagnostic criteria (8). Seven
first-degree relatives of the proband, ages 14–63 years, were studied.
All were free of symptoms of CAD and had normal resting
electrocardiograms. Nephrotic syndrome and hypothyroidism were
excluded. This study was approved by the ethics committee of the
Singapore General Hospital. Informed consent was obtained from all
subjects studied.
Blood was taken after a 10-h fast. Cholesterol and triglyceride
concentrations were measured by enzymatic methods using Kodak Ektachem
chemistry slides, which were then read on a Vitros 700 Chemistry
Analyzer. HDL-cholesterol was measured after
precipitation with dextran sulfate and magnesium chloride.
LDL-cholesterol (LDL-C) was calculated using Friedewald’s formula
(9). Only the pretreatment lipid profile is presented.
DNA was extracted from whole blood using phenol-chloroform extraction
and salt precipitation.
Mutations in the LDL-receptor gene were detected independently using
PCR-single strand conformation polymorphism (SSCP) analysis and
denaturing gradient gel electrophoresis (DGGE) in two laboratories.
pcr-sscp
Each PCR was performed in a final volume of 50 µL containing 0.2
µg of genomic DNA, 25 pmol of each oligonucleotide primer, 200 µmol
of deoxynucleotide triphosphates, and 1 U of Taq DNA
polymerase in the reaction buffer supplied (Promega). PCR was carried
out using the GeneAmp 9700 PCR system (Perkin-Elmer). The
oligonucleotide primers for the promoter region and the translated exon
sequences were as described previously (10). The PCR and
SSCP conditions are shown in Table 1
. After amplification, 5 µL of the PCR product was added to 5
µL of loading buffer (950 mL/L formamide, 0.5 g/L bromphenol
blue, 50 g/L EDTA), denatured by heating at 95 °C for 5 min, and
quenched on ice. SSCP analysis was carried out using the DCode
Universal Mutation Detection System (Bio-Rad). Samples were
electrophoresed in acrylamide gel without glycerol in Tris-borate-EDTA
(89 mmol/L Tris base, pH 8.3, 89 mmol/L boric acid, 2 mmol/L disodium
EDTA) at room temperature for 18–20 h at 120 V. The gel was stained
with silver nitrate (Silver Sequence DNA Sequencing System; Promega).
pcr-dgge
PCR amplification of the LDL-receptor gene was performed according
to Nissen et al. (11). Twenty pairs of GC-clamped primers
were used to amplify the promoter region and all 18 LDL-receptor exons,
including at least 5 intron bases on both sides of the exons (for exon
4, two sets of primers were designed for the 5' and 3' halves because
of the exceedingly long exon length). The standard PCR mixture for
LDL-receptor gene amplification in a total reaction volume of 25 µL
was as follows: 300 ng of genomic DNA, 50 pmol of each primer, 1.5 mM
MgCl2, 200 µmol of deoxynucleotide phosphates,
and 1 U of Taq DNA polymerase (Promega), in a reaction
buffer supplied by Promega. Deviations from the standard PCR mixture
were for the 3' end of exon 4 (1.0 mM MgCl2) and
for exon 15 (2.5 mM MgCl2 and 600 ng of genomic
DNA). The standardized thermal cycling profile consisted of an initial
denaturation step at 95 °C for 5 min, 40 cycles at 94 °C for 1
min and 66 °C for 5 min, and a final extension step at 72 °C for
10 min. This was then followed by a denaturation/renaturation program
at 99 °C for 7 min, 65 °C for 1 h, and 37 °C for 1
h, with a final soak at 4 °C, to generate hetero- and/or
homoduplexes from the amplified PCR products.
Parallel denaturing gradient gels of 20–60%, 30–70%, or 40–80%
denaturant (100% denaturant, 7 mol/L urea plus 400 mL/L
formamide) were made with a gradient gel mixer. In brief, amplified PCR
products from the apo B-100 gene were loaded onto 20–60% gels; PCR
products from exons 2, 3, 5, 6, 10, 11, 12, 13, and 17 and the promoter
region (LDL-receptor gene) were loaded onto 30–70% gels; and those
from the remaining LDL-receptor exons (1, 4–5', 4–3', 7, 8, 9, 14,
15, 16, and 18) were loaded onto 40–80% gels and then electrophoresed
for 6 h at 150 V.
Analysis of the apo B-100 gene used DGGE as described previously
(12). The primers used were
5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGGGAGCAGTTGACCACAAGCTTAGC-3'
with a 40-bp GC-clamp at the 5' end, and
5'-GGTGGCTTTGCTTGTTATGTTCTCC-3'. The putative LDL-receptor binding
region containing nucleotides 10551–10892 (corresponding to codons
3448–3562) of exon 26 of the apo B-100 gene was amplified and screened
for mutations.
All mutations found by SSCP or DGGE were confirmed by DNA sequencing.
For the LDL receptor, we used the ABI Prism 310 Genetic Analyzer
(Perkin-Elmer). Genomic DNA was amplified using the primers for the
exon in which SSCP or DGGE revealed a mutation. PCR conditions were in
accordance with the manufacturer’s recommendation. The product was
cleaned with QIAquick PCR purification reagent set (Qiagen)
before sequencing. The apo B-100 gene was sequenced using the same
primers without the GC-clamp as described previously (12).
apo E was genotyped by the method of Hixson and Vernier
(13).
LDL from the patients in this family was not available for studies to
assess the functional significance of the apo B-100 mutation found.
However, we were able to obtain material from 8 other patients with the
same mutation, and the functional significance of the mutation was
assessed by comparing the results of a U937 cell proliferation assay
(14)(15) in these 8 subjects against those of 10
healthy controls. Because Van den Broek et al. (15)
found that the cell density at the beginning of the experiment
influenced U937 cell proliferation, standardized conditions were
strictly followed in this study: (a) A fixed number of cells
(1 x 105 cells/mL) was used in all
experiments. (b) The cells were incubated in medium without
serum for 24 h to ensure intracellular cholesterol depletion.
(c) Frostegard et al. (14) observed that oxidized
LDL reduced the ability to stimulate U937 cell proliferation. Because
naturally occurring antioxidants in the d >1.21
kg/L fraction may prevent oxidation of the LDL, only pure LDL was used
for the experiments.
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Results
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The pedigree for the family is shown in Fig. 1
. A mutation in exon 9 of the LDL receptor was detected in the
proband (subject II-4) and one sibling (subject II-1) by both PCR-SSCP
(Fig. 2
) and PCR-DGGE analyses. Subsequent sequencing showed that this
was a C-to-G substitution at nucleotide position 1284
(Asp407Lys), a mutation that has previously
been shown to cause FH in South Africans (16). In addition,
a polymorphism in exon 10 (G-to-A substitution at nucleotide 1413) was
detected in both the proband and her sister (subject II-1). This has
been found to be common in healthy individuals (17). No
other abnormalities in the LDL-receptor gene were detected. Clinical
and biochemical characteristics of the family members are shown in
Table 2
. The father of the proband (subject I-2) suffered a sudden
death at the age of 45 years (20 years before the diagnosis of FH in
the proband), and no additional information was available for him.
Several members of the family (subjects I-1, II-3, II-4, II-5, III-1,
and III-2) had increased serum cholesterol (>7.5 mmol/L), which is
diagnostic of FH in first-degree relatives of a proband (8),
but did not carry the mutation. DGGE of the apo B-100 gene (Fig. 3
) showed that all but one of these subjects, i.e., the mother of
the proband (subject I-1), a brother (subject II-5), and two sons
(subjects III-1 and III-2) as well as the proband herself (subject
II-4), had the same mutation in the apo B-100 gene
(Arg3500Trp). The proband was thus a compound
heterozygote for both FH and FDB. All members of this family had the
33 wild-type genotype for apo E, which is not associated with
hypercholesterolemia. The hypercholesterolemia in subject II-3 is
currently unexplained. To rule out the possibility of an LDL-receptor
mutation that may have been present and undetected by SSCP or DGGE, we
sequenced all exons and the promoter region in this subject. No
additional abnormalities were detected.
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Figure 1. Pedigree for family reported.
The proband was a compound heterozygote for FH and FDB. Six of the
family members studied had hypercholesterolemia, and of these, five had
either FH or FDB.
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Figure 2. SSCP of exon 9 of the LDL receptor for the family members
studied demonstrating the abnormal pattern caused by a mutation in exon
9 (Asp407Lys).
Subjects are the same as in Fig. 1
.
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Figure 3. DGGE demonstrating an abnormal pattern in six members for
the family, including the index case, indicating a mutation in
the gene for apolipoprotein B-100 (Arg3500Trp)
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The results of the U937 cell proliferation assay are shown in Fig. 4
. The cell proliferation rate at 20 µmol/L LDL-C (mid-point of
this assay) was 0.77 for cells fed with LDL from the FDB subjects with
the Arg3500Trp mutation, compared with 4.01
for cells fed with LDL from healthy individuals, a fourfold reduction
in the cell proliferation rate. This trend was similar to those seen by
Gaffney et al. (18) and Van den Broek et al. (15)
with LDL from patients with the classical
Arg3500 Gln mutation.
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Figure 4. Results of the U937 cell proliferation assay.
U937 cells (1 x 105 cells/mL) were incubated in
medium without serum for 24 h to ensure intracellular cholesterol
depletion. Subsequently, LDL isolated from 8 patients with the
Arg3500Trp mutation in the apolipoprotein B-100 gene and from 10
control subjects was added to the culture medium in various
concentrations. Results are represented as means ± SD. The cell
proliferation rate at 20 µmol/L LDL-C (mid-point of this assay) was
0.77 for cells fed with LDL from the FDB subjects with the
Arg3500Trp mutation compared with 4.01 for cells fed
with LDL from healthy individuals.
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Discussion
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Exons 7–14 of the LDL-receptor gene encode the epidermal growth
factor precursor homology domain of the LDL-receptor protein,
consisting of 400 amino acids (19)(20). This
domain is required for the acid-dependent dissociation of the receptor
from its ligand during recycling (21). Thus, the amino acid
substitution, from asparagine to a positively charged lysine in exon 9,
found in some of our study subjects might interfere with receptor
recycling. The structure and function of this mutation have yet to be
studied.
When it was first described, FDB was thought to be produced by a single
mutation at codon 3500 of the apo B-100 gene
(22)(23). Since then, several other mutations
have been found, some of which were first described in an Asian
population (12). It was one of these mutations
(Arg3500Trp) that was found in this family. We
have now shown that this mutation leads to a loss of function similar
to that observed with the classical Arg3500Gln
mutation (Fig. 4
).
FDB represents a ligand-receptor disorder that is complementary to FH.
Compound heterozygotes with both FDB and FH are unusual, with only
three reports of compound heterozygotes in the literature
(5)(6)(7). Of these, only two groups characterized the
mutations by DNA analysis (6)(7). They found, as
we did, that compound heterozygotes exhibited more severe
hypercholesterolemia than did their first-degree relatives heterozygous
for either FH or FDB. The proband in this family had a pretreatment
serum LDL-C concentration almost twice as high as those of her siblings
(Table 2
). Such compound heterozygotes, we believe, may account for two
other phenomena occasionally observed in FH kindred. (a)
Families have been reported in which an LDL-receptor defect was not
found in all of the hypercholesterolemic first-degree relatives
of patients with FH (24). This was the case in this family,
and only DNA analysis of the apo B-100 gene allowed an accurate
diagnosis to be reached. The presence of a compound heterozygote in the
family must be considered in such cases and apo B-100 mutations sought
actively to avoid labeling an affected individual as healthy,
especially in children in whom the lipid profiles are less remarkable,
such as subjects III-1 and III-2. It is also necessary to look for
mutations other than the original Arg3500Gln
mutation because several other mutations are known to cause FDB in
Asians. (b) Some FH kindred, like the one presented here,
include individuals with exaggerated hypercholesterolemia compared with
other family members who have heterozygous FH. We should consider the
possibility that these individuals are compound heterozygotes for FH
and FDB.
Although the Asp407Lys mutation found in exon 9 of the
LDL receptor in the proband and in subject II-1 has been reported
previously as a cause of FH (16), we have not demonstrated
functional impairment of the LDL receptor in these individuals. The
unexplained hypercholesterolemia seen in subject II-3 (Table 2
) raises
the possibility that another, currently unidentified mutation may
contribute to the hypercholesterolemia seen in the proband as well as
in subjects II-1 and II-3. However, no other mutations were found in
the LDL receptors of our patients even with direct sequencing of the
entire coding region, and we believe that the exon 9 mutation we found
is significant. Of course, we cannot rule out the possibility that
genes at another locus could interact with mutations at the LDL
receptor or apo B-100 locus and may produce variations in phenotype in
affected family members. In the first reported family of this type, the
compound heterozygote was found to have lipid concentrations similar to
the concentrations in individuals with either defect (5).
The authors attributed this to other genetic or environmental factors
that may have been operating in this individual. Another example of
such gene-gene interaction is seen the recent work of Knoblauch et al.
(25), in which a locus on chromosome 13q was linked to
variations in cholesterol concentrations in a family with FH. Although
we agree that this deserves further investigation, we are unable to
speculate any further at this time.
Despite the exaggerated hypercholesterolemia, FH/FDB compound
heterozygotes retain an ability to respond to lipid-lowering therapy
(7). In our proband, treatment with 80 mg/day simvastatin
lowered LDL-C from 9.47 mmol/L to 4.82 mmol/L. This represents a 49%
reduction, which is similar to the 46% and 48% reductions in total
cholesterol seen in the two cases reported by Benlian et al.
(7).
When one genetic defect fails to explain the cause of
hypercholesterolemia in families who appear to have monogenic
hypercholesterolemia, the presence of both LDL receptor and apo B-100
defects should be considered.
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Acknowledgments
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This work was supported by the National Medical Research Council,
Singapore, Grant Number NMRC/0257/1997.
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Footnotes
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2 These authors contributed equally to this work. 
1 Nonstandard abbreviations: FH, familial hypercholesterolemia; CAD, coronary artery disease; FDB, familial defective apolipoprotein B-100; apo, apolipoprotein; LDL-C, LDL-cholesterol; SSCP, single strand conformation polymorphism; and DGGE, denaturing gradient gel electrophoresis.
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Abstract
Introduction
