© 1995 American Heart Association, Inc.
Articles |
FH Afrikaner-3 LDL Receptor Mutation Results in Defective LDL Receptors and Causes a Mild Form of Familial Hypercholesterolemia
From the Medical Research Council (MRC)/University of Cape Town Research Unit for the Cell Biology of Atherosclerosis (J.F.G. van R., D.R. van der W., G.A.C.), Department of Medical Biochemistry, and the Department of Medicine (A.D.M.), University of Cape Town, Cape Town, the MRC Division for Chronic Diseases of Lifestyle (K.S.), Tygerberg, and the Department of Human Genetics (E.L., M.J.K.), Faculty of Medicine, University of Stellenbosch, Tygerberg, South Africa.
Correspondence to D.R. van der Westhuyzen, UCT/MRC Research Unit for the Cell Biology of Atherosclerosis, Department of Medical Biochemistry, UCT Medical School, Observatory 7925, Cape Town, South Africa.
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Abstract Three founder-related gene mutations (FH Afrikaner-1, -2, and -3) that affect the LDL receptor are responsible for 90% of the familial hypercholesterolemia (FH) in South African Afrikaners. Patients heterozygous for the FH Afrikaner-1 (FH1) mutation, which results in receptors having
20% of normal receptor
activity, have significantly lower plasma cholesterol levels and milder
clinical symptoms than heterozygotes with the FH Afrikaner-2 mutation,
which completely abolishes LDL receptor activity. In this study we
re-created the FH3 mutation (Asp154
Asn) in exon 4 by
site-directed mutagenesis and analyzed the expression of the mutant
receptors in Chinese hamster ovary cells. The mutation resulted in the
formation of LDL receptors that are markedly defective in their ability
to bind LDL, whereas binding of apoE-containing ß-VLDL is less
affected. The mutant receptors are poorly expressed on the cell surface
as a result of significant degradation of receptor precursors. The
plasma cholesterol levels of 31 FH3 heterozygotes were similar to FH1
heterozygotes but significantly lower than FH2 heterozygotes. The FH1
and FH3 heterozygotes also tended to be less severely affected
clinically (by coronary heart disease and xanthomata) than FH2
patients. This study demonstrates that mutational
heterogeneity in the LDL receptor gene influences the
phenotypic expression of heterozygous FH and that severity of
expression correlates with the activity of the LDL receptor measured in
vitro. The results further indicate that knowledge of the specific
mutation underlying FH in heterozygotes is valuable in determining the
potential risk of premature atherosclerosis and should influence the
clinical management of FH patients.
Key Words: familial hypercholesterolemia • LDL receptor • founder • cholesterol
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Introduction |
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Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations at the LDL receptor locus.1 FH is one of the most common inherited disorders in humans, the frequency of heterozygous FH being approximately 1:500. The disorder is characterized clinically by elevated plasma LDL cholesterol (LDL-C), xanthomas, atheromas, and premature coronary heart disease (CHD). Homozygotes are particularly severely affected and often succumb to coronary disease before the age of 30 years. Nevertheless, phenotypic variation is encountered among homozygotes; those with receptor-defective alleles and residual receptor activity are less severely affected than patients with receptor-negative mutations.2 3 4 Patients who are heterozygous for FH also have a varied disease expression5 6 7 that could be due to a variety of genetic and nongenetic factors. The influence of other genes, such as apo(a),8 9 10 11 apoB,12 13 and apoE,7 14 15 16 17 has been investigated but does not provide a clear explanation for the phenotypic expression of FH. In a comparison of two different Afrikaner founder mutations, FH Afrikaner-1 (FH1) and -2 (FH2), we have shown that variability of LDL receptor mutations contributes to phenotypic variation in FH heterozygotes.18
FH is unusually prevalent (>1:100) in South African
Afrikaners.19 20 This is as a consequence of three
founder-type mutations in the LDL receptor.21 22 23 24 The FH1
and FH2 mutations correspond to receptor-defective and
receptor-negative alleles, respectively.25 The third
founder mutation, FH Afrikaner-3 (FH3; Asp154Asn,
GATAAT, in exon 4 of the LDL receptor gene) was found to be
associated with the disease22 but neither the consequence
of the mutation on receptor activity nor the variability of expression
in FH3 heterozygotes has been studied.
In the present study we have re-created the FH3 mutation by site-directed mutagenesis. We show that this mutation results in the formation of LDL receptors that are markedly defective in their ability to bind LDL, whereas receptor binding of the apoE-containing particle ß-VLDL is less affected. Furthermore, these receptors are poorly expressed on the cell surface as a result of a significant degree of degradation of receptor precursors. Analysis of the hypercholesterolemia and clinical features of patients heterozygous for the mutation indicates that the mutation is associated with a relatively mild form of FH, consistent with the defective ligand binding properties of the mutant receptor.
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Methods |
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Patients
A total of 31 Afrikaans-speaking unrelated patients, positive for the FH3 mutation, attended the lipid clinics at Tygerberg, Groote Schuur, and Johannesburg Hospitals. Blood was taken after obtaining informed consent and with ethical approval by the appropriate institution. Blood lipid values were determined on samples taken from fasting patients before any lipid-lowering treatment commenced. Patients were assessed for the presence or absence of tendon xanthomas and CHD (ie, myocardial infarction and/or angina pectoris).
Biochemical Determinations and Detection of FH3 Mutation and ApoE
Genotype
Plasma total triglycerides, total cholesterol (TC), and
HDL cholesterol (HDL-C) levels were determined,18 and
LDL-C levels were calculated according to the Friedewald-Levy
formula.
Genomic DNA was extracted from peripheral blood samples. The point
mutation at codon 154 was detected by Mbo II digestion of
polymerase chain reaction (PCR)–amplified fragments.22
Apo 
genotypes were determined by PCR amplification of
genomic DNA and Hha I digestion according to Hixson and
Vernier.26
Lipoprotein and Antibody Preparations
LDL (d=1.019 to 1.063 g/mL) and lipoprotein-deficient
serum (LPDS) (d>1.25 g/mL) were prepared from human
plasma.27 ß-VLDL was prepared from the plasma of
cholesterol-fed rabbits as described by Kovanen et al.28
The lipoproteins were iodinated using the iodine
monochloride method.27 The monoclonal antibody IgG-C7,
directed against the first repeat in the binding domain of the LDL
receptor, was prepared from hybridoma cells obtained from the American
Type Culture Collection (CRL/691) and was iodinated by
using Iodo-Gen (Pierce Chemical) according to the method of Beisiegel
et al.29
Cells and LDL Receptor Assays
Skin fibroblasts were obtained from an LDL receptor–normal
person and from an FH3 heterozygote patient. The cell line, ldlA7 (a
mutant Chinese hamster ovary [CHO] cell line lacking functional LDL
receptors) was transfected with a plasmid expressing the FH3 mutant LDL
receptor. Fibroblasts and CHO cells were cultured.30
Binding and biosynthetic studies in fibroblasts were performed on cells
in which receptor activity was upregulated by a 48-hour incubation in
LPDS.
Surface binding at 4°C and metabolism at 37°C of labeled lipoproteins and antibody were performed.30 Nonlinear regression analyses using the program ENZFITTER (Elsevier/Biosoft) were used to calculate Kd values and to obtain high-affinity components of ligand concentration curves assuming ligand binding to a single class of high-affinity sites and a nonspecific process. For biosynthetic studies, cells were incubated with [35S]methionine (±100 µCi/mL) in methionine-free medium and chased in medium containing 200 µmol/L unlabeled methionine. LDL receptors were solubilized, immunoprecipitated by using IgG-C7/goat anti-mouse IgG immune complexes, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and visualized by fluorography.31
Site-Directed Mutagenesis
Oligonucleotide-directed mutagenesis using single-stranded M13
DNA as template was performed as described by Kunkel.32 An
Xba I–EcoRI 754-bp restriction fragment from
pLDL receptor 2 (exons 1 through 4)33 was subcloned into
the multiple cloning site of M13BM21RF34 and grown in
JM109 Escherichia coli cells. A
HincII-EcoRI 480-bp restriction fragment (3' half
of exons 3 through 4) was subcloned from the Xba
I–EcoRI insert into M13BM21RF. Single-stranded DNA was
isolated and used as a template for the mutagenesis reaction. The
mutagenic oligonucleotide (5'-CCGAGCCATTTTCGCAGT-3') was an
18 mer with eight and nine perfectly matched nucleotides on
either side of the mismatch (T/G). The entire 480-bp mutagenized
fragment was sequenced to exclude the possibility of errors at other
sites and subcloned back into the parent 754-bp Xba
I–EcoRI fragment in M13BM21RF. The Xba
I–EcoRI fragment was cloned into pLDL receptor 2. The
plasmid was transfected into ldlA7 cells together with pSVneo by using
the calcium phosphate precipitation technique, and a stable cell line
was established as described by Davis et al.35
Statistical Methods
The data from the participating centers were analyzed with the
aid of a statistical software package (INSTAT, Graphpad
Software). ANOVA was used to analyze parametric data, deriving
two-tailed q values. In the comparison of TC in the groups
without and with tendon xanthomata, the data were transformed to the
logarithmic value because the distribution did not follow a gaussian
pattern. The correlations of lipids with age or gender were assessed by
linear regression, and the r2 value was
used as a guide to the contribution the parameter made to the
correlation.
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Results |
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To determine whether the FH3 mutation results in receptors that are functionally defective, the mutation was introduced into the coding region of the LDL receptor gene by site-directed mutagenesis, and the mutant receptor was then studied in transfected CHO cells. The ability of the mutant receptors to bind LDL and ß-VLDL was investigated. To account for variability in the degree of expression of the transfected genes, ligand binding values are expressed in terms of the binding of a monoclonal anti-receptor antibody (IgG-C7). This antibody bound to the mutant FH3 receptor with normal high affinity (data not shown). At 4°C, the binding of 125I-LDL to the FH3 receptor was severely impaired compared with the normal receptor transfected in the same manner (Fig 1
). The affinity of the mutant
receptors for LDL was approximately two times lower than normal, while
maximal binding at ligand saturation was only about 20% of normal.
These results indicated that only a small fraction of the mutant
receptors appeared to bind LDL and that this binding occurred with a
reduced affinity. Since maximal LDL binding normalized to antibody
binding was only 20% of normal, it also followed that a large
proportion of FH3 receptors was unable to bind LDL at all.
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The ability of the mutant receptor to bind to apoE was assessed by
using apoE-containing ß-VLDL obtained from rabbits. Rabbit and human
apoE share an 80% homology in amino acid sequence, most key residues
are identical, and the LDL receptor binding sequence (residues 136
through 150 in human apoE) is identical in the two
proteins.36 The two proteins exhibit similar high-binding
affinity to the LDL receptor when presented in proteolipid
complexes.37 In contrast to the binding of LDL, the
binding of ß-VLDL was far less severely affected (Fig 1
). All
immunoreactive receptors bound ß-VLDL as indicated by the binding at
ligand saturation, and the affinity of the mutant receptors for
ß-VLDL was about two- to threefold lower than that of normal
receptors.
The impaired ability of mutant FH3 receptors to bind LDL accounted for
the decreased receptor-mediated uptake and degradation of LDL at 37°C
observed in the CHO cells expressing the FH3 receptors. LDL degradation
by the FH3 receptors was 20% of normal at ligand saturation (data
not shown). The uptake and degradation of ß-VLDL by the mutant
receptors, as in the case of its binding measured at 4°C, were
significantly less affected than the uptake and degradation of LDL.
Maximal ß-VLDL degradation by the FH3 receptors was 75% of normal
(data not shown). The reduction (25%) in ß-VLDL degradation relative
to its binding by the mutant receptor implies a certain deficiency in
receptor internalization or recycling, but this possibility was not
investigated further in this study.
To investigate the possible effects of the mutation on receptor
biosynthesis, processing, and turnover, the metabolism of
biosynthetically labeled receptors was studied in the transfected CHO
cells. The mutant FH3 receptors were synthesized as a normal-sized
120-kD precursor (Fig 2). However, in comparison to
normal receptors, the processing of mutant precursors to the mature
160-kD form of the receptor was markedly retarded. No mature form of
the mutant receptor was visible following a 1-hour chase period. In
contrast, the normal receptor was completely processed to the mature
160-kD form within 1 hour. The retarded processing of the mutant
precursors was accompanied by a significant loss of receptor, such that
only about 20% of the initial precursors were recovered as mature
receptors after an 8-hour chase.
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To determine if the decreased expression of mature FH3 receptors could
at least be partly accounted for by a decreased stability of mature
receptors, the half-life of the mature FH3 receptor was determined and
compared with that of the normal receptor. LDL receptors were found to
be degraded by a process that followed first-order kinetics (Fig 3).25 38 The half-life of the mutant
receptor (10 hours) was only slightly shorter than the half-life of the
normal receptor (14 hours). The FH3 mutation therefore does not
markedly influence the stability of the mature form of the receptor.
The low cellular expression of the mature FH3 receptor is thus largely
due to retarded precursor processing and the accompanying breakdown of
a large proportion of receptor precursors.
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values were 14.4 and 10.3 hours for the normal
and mutant LDL receptors, respectively.
No patients who are true homozygotes for the FH3 mutation have yet been
identified. It is not possible to accurately assess the binding
properties of this mutant receptor in cells obtained from heterozygous
FH patients owing to the presence of normal receptors expressed from
the normal allele in these patients. However, biosynthetic studies in
fibroblasts cultured from a patient heterozygous for the mutation
yielded results that were completely consistent with the findings
obtained with the transfected CHO-FH3 cells. In contrast to LDL
receptors in normal fibroblasts, which were completely processed to the
mature form during a 1-hour chase period, about half the receptor
precursors in the patient's cells remained unprocessed after this
chase period (Fig 4). Even after a 3-hour chase period
some receptors remained in the precursor form. As described above, such
slow processing was characteristic of the FH3 receptor in CHO
cells.
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A total of 31 unrelated FH heterozygotes with the FH3 mutation were
identified, and the lipid and lipoprotein levels of these individuals
were analyzed (Table 1). These were compared with our
earlier values for normal Afrikaners18 as well as for FH1
and FH2 heterozygotes. The mean TC and LDL-C levels of the FH3 group
were clearly elevated compared with the normal group, whereas the mean
HDL-C and triglyceride levels were higher in the control subject group.
No significant differences in TC, LDL-C, or other levels were observed
between the FH3 and FH1 groups. On the other hand, significant
differences in TC and LDL-C levels were observed between the FH2 and
FH3 groups as well as between the FH2 and FH1 groups.
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The contribution of certain variables to the variation in TC within the FH3 group was determined by univariate analysis. Smoking status, HDL-C levels, and gender were noncontributory, but variation in triglyceride (21%) and age (14%) did contribute to the variation found in TC.
The possible contribution of apoE polymorphism to the variation in
TC between the FH3 and FH2 groups was also considered. Of the 31
heterozygotes within the FH3 group, 26 had an 3/3 genotype,
and no 2 alleles were present (data not shown). Most
importantly, when only the 3/3 patients of each group were compared,
mean TC levels remained significantly different between the two groups.
In contrast, no difference was observed between the FH3 and FH1
groups.
The LDL-C levels in the unrelated FH3 heterozygotes with and without
CHD and xanthomata were plotted against patient age (Fig 5). The frequency of both CHD and xanthomata clearly
increased with age. No CHD was observed in any of the patients who were
under 30 years of age, whereas the frequency of CHD and xanthomata in
patients over 45 years of age reached 50% and 86%, respectively.
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The lipid and lipoprotein concentrations of the FH3 heterozygotes
between 20 and 50 years of age are shown in Table 2.
Within this age group, 20% of patients had CHD, and 39% had tendinous
xanthomata. These values are similar to those for FH1
heterozygotes.18 Comparative values for the FH2 group were
43% with CHD and 67% with xanthomata. Although none of these
differences in the clinical manifestations between FH3 and FH2 subjects
reached statistical significance in these small groups, they
suggest a tendency for the FH3 heterozygotes to be less severely
affected clinically than the FH2 heterozygotes. Within the FH3 group,
no significant difference in TC or LDL-C was observed between the CHD
and non-CHD patients, even though the mean age of the CHD group was
higher than the non-CHD group (Table 2). On the other hand, a
significant difference was observed in TC and LDL-C in the same group
between patients with and without xanthomata.
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Discussion |
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FH3 is the least prevalent of the Afrikaner founder mutations, accounting for about 5% to 10% of FH in this population.23 24 While this mutation has a lower frequency than the FH1 and FH2 mutations, it has been generally referred to as a founder mutation since its frequency is far greater than the individual frequencies of the many other known LDL receptor mutations (>150) found in other populations. Evidence that this mutation causes FH was originally supplied by restriction fragment length polymorphism–haplotype and segregation studies in affected families.22 39 40 Genealogical studies have traced the possible introduction of this defective gene into South Africa back to 1692.41 The minor role played by the FH3 gene in Afrikaners might be explained by the fact that the initial immigrant, possibly of English descent,42 had only three children, of whom only one presumably transmitted the defective FH3 gene to successive generations.
To confirm that this mutation causes FH and that it is not simply a silent polymorphism, we re-created the mutation by site-directed mutagenesis and studied the expression of the mutant receptor in transfected cells. The mutation markedly affected LDL receptor expression in two ways: by causing significant degradation of receptor precursors and by affecting the ligand-binding properties of the mature surface receptors. The FH3 mutation is therefore of a receptor-defective type, giving rise to low numbers of receptors that exhibit very low LDL binding but almost normal ß-VLDL binding.
The reduced number of mutant FH3 receptors on the cell surface is associated with the slow transport of receptor precursors from the endoplasmic reticulum (ER) to the Golgi apparatus and a significant degradation of these precursors. In contrast to its precursor, the mature form of the FH3 receptor exhibits the same stability as the normal receptor. Precursor retardation in the ER is typical of LDL receptor mutations designated class 2 mutations that typically, like the FH3 mutation, affect the ligand-binding domain of the receptor.43 This domain is composed of cysteine-rich repeats that are evidently altered conformationally in the class 2 mutants, resulting in their retention in the ER. The mechanisms involved in this retention and in the subsequent degradation of receptor precursors are not known. Chaperonin-type proteins have been implicated in the processing of other proteins in the ER,44 and an ER degradation system may possibly be responsible for the breakdown of FH3 precursor molecules.45 Some class 2 mutant receptors are subject to complete and rapid degradation in the ER.43 In others, including the FH3 mutant, a significant fraction of precursors is apparently normally glycosylated and transported to the cell surface. In the case of the FH1 mutation, most precursors reach the cell surface despite undergoing markedly delayed processing in the ER.30
The defective ligand-binding properties of the mutant FH3 LDL receptor
are characteristic of a class 3 LDL receptor mutation. The FH3 single
amino acid substitution (Asp154Asn) lies in the fourth
cysteine-rich repeat of the ligand-binding domain within a highly
conserved motif that appears to be essential for LDL
binding.46 Mutant FH3 receptors were observed to have
20% of the binding capacity of normal receptors for LDL. The
relative rates of uptake and degradation of LDL paralleled ligand
binding, indicating that these properties were not affected by the
mutation. Interestingly, this reduced binding of LDL was not due to a
reduction in binding affinity but was largely the result of a reduction
in binding capacity. The explanation for this is not clear, although we
have proposed that in the case of the mutant FH1 receptor, which
exhibits certain similar binding characteristics to the FH3 receptor,
the mutant surface receptors are present in two different forms,
one that binds LDL with normal high affinity and one that does
not.30 Similarly, our results on the FH3 mutation indicate
that only a small proportion of mutant receptors are capable of binding
LDL.
In contrast to the binding of LDL, the binding of the apoE-containing ligand ß-VLDL to the FH3 receptors was less affected. At saturating ligand concentrations, mutant and normal receptors bound approximately equal amounts of ligand. This feature agrees with observations that repeat 5 of the binding domain is critical for apoE binding, whereas mutations in any of the other repeats, such as repeat 4, which is altered in FH3, are more easily tolerated.47 The ability of the mutant FH3 receptors to bind apoE of ß-VLDL raises the possibility that the clearance of apoE-containing precursors of LDL is relatively normal in FH3 heterozygotes. This in turn would allow for a normal rate of LDL production compared with the situation in which the failure to clear LDL precursors results in an increased rate of LDL production.48 An increased rate of LDL production together with a decreased rate of LDL clearance would contribute to an increase in plasma LDL level.
The phenotypic expression of the FH3 mutation was analyzed in 31
unrelated Afrikaner FH heterozygotes. Plasma cholesterol levels
increased with age, which contributed 14% and 16% to the variation in
TC and LDL-C, respectively. This is similar to the age-dependent
increase in plasma cholesterol observed in normal subjects and in other
groups of FH heterozygotes.7 No significant difference in
cholesterol was observed between men and women, nor did HDL-C and
smoking contribute to the variation in cholesterol levels. In terms of
a possible effect of apoE isoforms, the large majority of patients were
of an 3/3 genotype, and it was not possible to analyze the
effects of different apoE alleles on cholesterol levels in these
patients.
The plasma lipid levels in the FH3 group of patients were compared with those in two other heterozygote groups, ie, the FH1 and FH2 groups. Both TC and LDL-C levels in the FH3 group were similar to those in the receptor-defective FH1 group, whereas they were significantly lower than the levels found in the receptor-negative FH2 group. Cholesterol levels in these heterozygotes are therefore consistent with the mutation type, the two receptor-defective groups (FH1 and FH3) giving rise to lower levels than the receptor-negative FH2 mutation. A similar difference between receptor-defective and receptor-negative mutations has been reported in patients homozygous for FH.2 3 4 The lower cholesterol levels in the defective groups, observed here even in the heterozygous condition, could be due to the residual low but significant activity expressed by these receptors. As discussed above, the rate of production of LDL in FH3 heterozygotes also may be less elevated than in receptor-negative heterozygotes owing to the ability of the mutant receptor to bind certain apoE-containing lipoproteins.
Similar to the other receptor-defective group (FH1), the FH3 heterozygotes show a tendency to be less severely affected clinically than the receptor-negative FH2 group. However, the possibility that the plasma LDL-C concentration may account for the clinical differences observed between the different mutation types could not be determined owing to the small number of patients. Within the FH3 group, no correlation was evident between CHD and cholesterol levels. It is likely that patients who had previously been treated for ischemic heart disease presented to the clinic after dietary modification, which could obscure the trend. On the other hand, a correlation was shown between cholesterol levels and the presence of xanthomata.
Our results therefore indicate that patients heterozygous for the FH3 receptor-defective mutation are affected relatively mildly biochemically compared with individuals carrying a receptor-negative type of mutation. We also confirm our previous study, demonstrating for the first time that mutational heterogeneity in the LDL receptor gene influences the phenotypic expression of heterozygous FH. The identification and knowledge of the specific mutation underlying FH in heterozygotes is therefore of value in determining the potential risk of premature atherosclerosis.
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Acknowledgments |
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This work was supported by the University of Cape Town, the South African Medical Research Council, the Cape Provincial Administration, and a donation from the Old Mutual. H. Karjiker, S. Jones, and M. Moodie are thanked for technical assistance, Dr J. Firth for some of the clinical details about the patients, and the nursing staff of Genetic Services, Department of National Health and Population Development, for the collection of blood samples.
Received March 30, 1994; accepted March 3, 1995.
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