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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:765-772

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:765-772.)
© 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

J.F. Graadt van Roggen; D.R. van der Westhuyzen; G.A. Coetzee; A.D. Marais; K. Steyn; E. Langenhoven; M.J. Kotze

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.


*    Abstract
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 *Abstract
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 down arrowResults
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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 {approx}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


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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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; Asp154->Asn, GAT->AAT, 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.


*    Methods
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 up arrowIntroduction
 *Methods
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 down arrowDiscussion
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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 {varepsilon} 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.


*    Results
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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 1Down). 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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  		Figure 1. Line graphs showing surface binding at 4°C of (top) 125I-LDL and (bottom) 125I–ß-VLDL to Chinese hamster ovary (CHO) cells transfected with normal (CHO-N) or familial hypercholesterolemia Afrikaner-3 mutant (CHO-FH3) LDL receptor. Cells were incubated with the indicated concentrations of 125I-LDL, 125I–ß-VLDL, or 125I–IgG-C7 (1 µg/mL) for 2 hours at 4°C. The data points (average of duplicates), which represent high-affinity binding values, are expressed as nanograms ligand bound per nanogram IgG-C7 bound and are calculated by nonlinear regression analysis of total binding curves. The calculated Kd values for 125I-LDL were 0.85 and 1.6 µg/mL for CHO-N and CHO-FH3 cells, respectively. Kd values for 125I–ß-VLDL were 0.17 and 0.6 µg/mL for CHO-N and CHO-FH3 cells, respectively. Maximum high-affinity binding of 125I-LDL in CHO-N and CHO-FH3 cells was 3.8 and 0.68 (20% of normal) ng/ng 125I–IgG-C7, respectively. Maximum high-affinity binding of 125I–ß-VLDL in CHO-N and CHO-FH3 cells was 2.2 and 2.3 (105% of normal) ng/ng 125I–IgG-C7, respectively. Similar values were obtained in two other experiments.

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 1Up). 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 {approx}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 {approx}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 2Down). 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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  		Figure 2. Biosynthesis and processing of 35S-labeled LDL receptors in Chinese hamster ovary (CHO) cells transfected with normal (CHO-N) or familial hypercholesterolemia Afrikaner-3 mutant (CHO-FH3) LDL receptor. Confluent CHO-N and CHO-FH3 monolayers were preincubated with methionine-free minimum essential medium and lipoprotein-deficient serum (LPDS) for 30 minutes and then pulse-labeled with [35S]methionine (100 µCi/mL) for 1.5 hours at 37°C. Cells were chased in minimal essential medium/LPDS for the indicated times using duplicate dishes. Cells were solubilized, and LDL receptors were immunoprecipitated and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and fluorography.

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 3Down).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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  		Figure 3. Degradation of 35S-labeled LDL receptors in Chinese hamster ovary (CHO) cells transfected with normal (CHO-N) or familial hypercholesterolemia Afrikaner-3 mutant (CHO-FH3) LDL receptor. Confluent CHO-N and CHO-FH3 cells were pulse-labeled for 1.75 hours at 37°C with [35S]methionine (63 µCi/mL). CHO-N and CHO-FH3 cells were then incubated in minimum essential medium and lipoprotein-deficient serum for 1 and 10.5 hours, respectively, to ensure that no 35S-label remained in the LDL receptor precursor pools. Cells were chased for the indicated times using duplicate dishes. Cells were then solubilized, and LDL receptors were immunoprecipitated and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and fluorography. The t 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 4Down). 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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  		Figure 4. Biosynthesis and processing of 35S-labeled LDL receptors in fibroblasts from a normal subject (N) and a subject heterozygous for the familial hypercholesterolemia Afrikaner-3 (FH) mutation. Fibroblasts were incubated for 48 hours in lipoprotein-deficient medium and then preincubated for 30 minutes at 37°C in methionine-free minimum essential medium. Cells were pulsed for 1 hour with [35S]methionine (100 µCi/mL) and then chased for the indicated times. Cells were solubilized, and LDL receptors were immunoprecipitated and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and fluorography.

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 1Down). 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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  		Table 1. Lipid and Lipoprotein Concentrations of Unrelated Persons

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 {varepsilon}3/3 genotype, and no {varepsilon}2 alleles were present (data not shown). Most importantly, when only the {varepsilon}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 5Down). 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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  		Figure 5. Scatterplots of LDL cholesterol (LDLC) levels according to age in subjects heterozygous for the familial hypercholesterolemia Afrikaner-3 mutation (FH3). Circles indicate probands with and squares probands without coronary heart disease (CHD) or tendinous xanthomata (XMTA).

The lipid and lipoprotein concentrations of the FH3 heterozygotes between 20 and 50 years of age are shown in Table 2Down. 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 2Down). 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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  		Table 2. Lipid and Lipoprotein Concentrations in FH3 Probands Aged 20 Through 50 Years


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
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 (Asp154->Asn) 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 {approx}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 {varepsilon}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.


*    Acknowledgments
 
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.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriven CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. 6th ed.New York, NY: McGraw-Hill Book Co; 1989:1195-1250.
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  4. Moorjani S, Roy M, Torres A, Betard C, Gagne C, Lambert M, Brun D, Davignon J, Lupien P. Mutations of low-density-lipoprotein-receptor gene, variation in plasma cholesterol, and expression of coronary heart disease in homozygous familial hypercholesterolaemia. Lancet. 1993;341:1303-1306. [Medline] [Order article via Infotrieve]
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