© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
R3531C Mutation in the Apolipoprotein B Gene Is Not Sufficient to Cause Hypercholesterolemia
From INSERM U383, the Hôpital Necker-Enfants Malades, Paris, France (J.-P.R., M.V., M.D., L.V., C.J., C.B.), Laboratoire de Biochimie et de Génétique Moléculaire (J.-P.R., C.J., C.B.) and Laboratoire de Biostatistique et d’Informatique Médicale (P.A.), Hôpital A. Paré, Boulogne, France, and Service d’Endocrinologie et de Nutrition, Hôpital Hôtel-Dieu, Nantes, France (M.K.).
Correspondence to C. Boileau, INSERM U383, Hôpital Necker-Enfants Malades, 149-161, rue de Sèvres 75743, Paris, Cedex 15, France. E-mail boileau{at}necker.fr
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Abstract—Familial hypercholesterolemia and familial ligand-defective apolipoprotein B-100 (FDB) are dominantly inherited disorders leading to impaired low-density lipoprotein receptor (LDLR) and apolipoprotein B-100 (APOB) interaction, plasma LDL elevation, and hypercholesterolemia. We previously identified the first French FDB-R3531C proband, a woman with very high total cholesterol, in a group of type IIa hypercholesterolemic families. We report here the investigation of her family at large that revealed the total absence of cosegregation with hypercholesterolemia. Six of the 10 subjects heterozygous for the R3531C mutation had plasma cholesterol lower than the 97.5th percentile for their age and gender, and mean cholesterol levels were not significantly different between affected and unaffected persons. Furthermore, 2 family members with similar high LDL-cholesterol levels were not carriers of the R3531C substitution, suggesting the implication of another mutation. Segregation analysis of the LDLR gene revealed statistically significant genetic linkage with hypercholesterolemia, and analysis of the proband LDLR gene led to the identification of the 664 proline to leucine defective mutation and its detection in all 6 hypercholesterolemic-related members of this family. Therefore, our results show that the family presents with familial hypercholesterolemia and give evidence that the R3531C substitution in the APOB gene is not an allelic variant leading to FDB. Furthermore, thorough analysis of our data suggests that the APOB-R3531C mutation enhances the hypercholesterolemic effect of the LDLR-P664L defect, suggesting that it is a susceptibility mutation.
Key Words: hypercholesterolemia • apolipoprotein B • familial ligand-defective apolipoprotein B • low-density lipoprotein • familial hypercholesterolemia.
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Introduction |
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Dominantly inherited type IIa dyslipoproteinemia is genetically heterogeneous and involves mutations in at least 3 genes: the gene encoding the low-density lipoprotein receptor (LDLR), the gene encoding its ligand apolipoprotein B-100 (APOB), and a recently mapped but still unidentified gene called FH3 at 1p34.1-p32.1 More than 600 mutations have been identified in the LDLR gene2 that lead to classical familial hypercholesterolemia (FH).3 Conversely, only 3 mutations have been identified in the APOB gene that induce familial ligand-defective apolipoprotein B-100 (FDB) by failure of LDL binding to its receptor and secondary plasma LDL-cholesterol elevation. The Arg3500
Gln mutation (R3500Q) was the first
established cause of FDB,4 and its frequency has been
estimated as 0.4% in Switzerland5 and 0.08% in North
Europe and United States.6 7 8 Binding affinity of LDL from
FDB-R3500Q heterozygotes is 36% that of normal.9 Clinical
features and total and LDL-cholesterol levels in most
FDB-R3500Q patients are often less pronounced than those observed in FH
patients.10 11 12 A second mutation was identified at codon
3500 (an ArgTrp substitution) in 13 probands: 12 of Asian and 1 of
Scottish origin.13 14 15 In an LDL functional assay using
the cell line U937, which has an absolute requirement for extracellular
LDL-cholesterol for growth, Gaffney et al13
showed that the relative growth rates of the cells were comparable and
as low (50% compared with control LDL) with LDL particles from
FDB-R3500Q subjects or FDB-R3500W subjects. This result suggested that
the binding affinity of the 2 groups of mutation-carrying LDL particles
was comparable. Finally, an ArgCys substitution was reported at
residue 3531 (APOB-R3531C), and binding affinity of LDL from FDB-R3531C
heterozygotes was between 63% and 70% that of
normal.9 16 17 Although numerous R3500Q mutations have
been reported in the United States and Europe, to date the R3531C
substitution in the APOB gene has only been reported in 27 probands
from the white population (Table
I).6 9 16 18 19 20 Four
were identified in unselected volunteers20 and 7 in the
general population of Denmark, leading to a frequency of
0.08%.6 Sixteen probands were identified in patients with
hyperlipidemia9 18 19 20
(0.15%9 to 0.5%18 ) or
coronary artery disease (CAD)6 16 19
(0.1%6 19 to 0.8%16 ). We report here the
analysis of the large family of 1
hypercholesterolemic proband.19
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Methods |
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Subjects and Blood Samples
Blood samples were collected after an overnight fast from 22 members of the HC5 family. Informed consent was obtained from all family members. Blood was either anticoagulated with potassium-EDTA or allowed to clot and the serum separated by centrifugation.
Serum Lipid Analysis
Serum triglycerides, total cholesterol,
and high-density lipoprotein cholesterol were
analyzed by standard enzymatic methods, either by direct assay
or after phosphotungstate and magnesium precipitation in the case of
high-density lipoprotein particles. LDL-cholesterol was
indirectly determined using the Friedewald formula.21
DNA Studies
APOB Gene
DNA was isolated from whole blood,22 and the R3500Q
and R3531C mutations were detected simultaneously using the
PCR-mediated site-directed mutagenesis method we described
previously.19 Ten APOB gene markers were analyzed,
as reported by Loux et al23: 8 biallelic markers
(insertion or deletion polymorphism in the signal peptide,
ApaLI, HincII, PvuII, AluI,
XbaI, MspI, and EcoRI) and 2
multiallelic markers (the 5' TGn marker and 3'HVR).
LDLR Gene
Two LDLR locus polymorphisms were studied: D19S394 and
D19S221.24 These multiallelic markers lie, respectively,
250-kilobase telomeric and 1-megabase centromeric to the LDLR gene.
Their heterozygosity indices are 0.9 and 0.8, respectively. For the
proband, each exon of the LDLR gene was amplified using specific
oligonucleotides, as described by Leitersdof et
al,25 and sequenced directly by the dideoxy method. The
detection of the P664-L mutation in family members included
amplification of exon 14, digestion with PstI,
electrophoresis through a 6% acrylamide gel, and
visualization with ethidium bromide.26
Apolipoprotein E Gene
Apolipoprotein E genotype was determined using the
INNO-LiPA Apo E kit (Innogenetics) as recommended by the
manufacturer.
Linkage Analysis
Pairwise and multipoint linkage analyses were performed
using the SLINK and MLINK programs of the LINKAGE
package.27 Lod scores were computed with a complete and
incomplete (0.9) penetrance of the trait defined as plasma
cholesterol above the 97.5th percentile28 for
heterozygous individuals and with a gene frequency of 0.002. Linkage
was investigated assuming equal female to male recombination rates.
Statistical Analysis
Plasma levels of total cholesterol were expressed as
multiple of median (MoM) for age and gender using the reference for the
French population.28 Plasma levels of
LDL-cholesterol were not adjusted for age and gender,
because no French population data have been published for this
parameter. Data are expressed as mean±95% confidence
interval. The Statistical Package for the Social Sciences, version 8.0
for Windows, was used for the statistical analysis. We
performed a 2-way ANOVA to test the effect of the APOB-R3531C and
LDLR-P664L mutations on the phenotype in carriers identified in
the family and tested the interaction between the 2 factors.
P<0.05 was considered significant.
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Results |
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Nuclear Family Study
The family (Figure
I) was
investigated through the mother (II-2), who presented bilateral
corneal arcus associated with highly elevated total and
LDL-cholesterol levels poorly lowered by treatment with
3-hydroxy 3-methylglutaryl coenzyme A reductase inhibitors.
Ultrasound investigation revealed atherosclerosis in
the carotid arteries. Her 13-year-old son (III-1) also displayed highly
elevated total and LDL-cholesterol levels requiring
lipid-lowering therapy with bile-acid sequestrants. Serum
cholesterol levels (Table
II) were not high (clearly below
the 97.5th percentile) for her daughter (III-2) and husband (II-1) when
compared with gender- and age-matched levels established in the French
population.28
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To identify the molecular defect associated with the
hypercholesterolemic phenotype observed in the
family, we first screened for the 2 APOB mutations in the white
population (OMIM *107730.0017 and 0009) leading to FDB.17
No R3500Q mutation was found, but the R3531C mutation of the APOB gene
was found to be heterozygous in the mother and her 2 children.
Surprisingly, the same mutation was also identified in the father.
Family members were separately resampled and retested to eliminate the
possibility of incorrect sample assignment. The presence of the
mutation was confirmed in all subjects. Genotypes were
determined for 10 APOB polymorphic markers, and complete haplotypes
could be deduced unequivocally for the proband, her husband, and their
2 children (Figure I). The mutation in the proband (II-2) and
her husband (II-1) was associated with haplotype E as in the Celtic and
Native American kindred originally reported by Pullinger et
al9 and in the 2 index cases of Celtic origin reported by
Wenham et al.18 This result suggested that the parents
(II-1 and II-2), both from the western part of France, were of hitherto
unknown common Celtic descent. The APOB haplotype analysis in
this nuclear family also revealed that the asymptomatic
daughter had inherited the mutation from her
hypercholesterolemic mother, whereas her
hypercholesterolemic brother had inherited the mutation
from his normocholesterolemic father. Because the
discrepancy between the molecular and laboratory data could be
attributed either to a lack of penetrance of the R3531C mutation or the
existence of a defect in another major gene, investigation of the
family at large was undertaken.
Study of the Family at Large
Eighteen additional subjects were available for study (Figure
II). Serum lipid levels were determined
for all subjects (Table II), and DNA analysis was
performed. Six additional heterozygous carriers of the R3531C mutation
were identified (Figure II). The proband’s husband (II-1)
inherited the mutation from his asymptomatic mother (I-2).
Furthermore, his family history revealed no instances of
hypercholesterolemia or CAD. Conversely, the
father (I-3) of the proband (II-2) had documented
hypercholesterolemia, as did 2 of the
proband’s siblings (II-6 and II-9) and 2 of her nephews (III-6 and
III-8). Despite the presence in her family of elevated lipid values and
the R3531C mutation, there was no segregation of the mutation with the
very high total cholesterol levels (Figure II).
Linkage was excluded on the basis of lod scores equal to -2.64 and
-2.61 with complete and incomplete penetrance, respectively.
Furthermore, there was no significant difference in lipid levels
between carriers of the R3531C mutation (mean value and 95% confidence
interval for total cholesterol and
LDL-cholesterol were 6.77 [5.48 to 8.06] and 4.34 [3.21
to 5.48] mmol/L, respectively) and family members lacking the
mutation (6.02 [5.31 to 6.72] and 4.13 [3.47 to 4.8] mmol/L,
respectively). Variations between different individuals in the
expression of an identical mutation is well documented in dominantly
inherited disorders and has been reported for the common FDB mutation
R3500Q.29 30 These variations may be explained by
differences in environmental factors but are unlikely in a single
family. Furthermore, 2 subjects, a woman (II-9) and her son (III-8),
with severe hypercholesterolemia did not carry
the R3531C mutation, suggesting the existence of a second mutation in
the maternal family.
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Using the SLINK program and assuming a marker with a heterozygosity
index of 0.83 (very close to the heterozygosity index of the 2 LDLR
markers used in the study), we estimated expected maximum lod scores of
2.36 with complete penetrance and 2.24 with incomplete penetrance.
These scores are above the threshold level (lod score=2) that is
statistically significant for linkage when analyzing a candidate
gene.31 Therefore, because the family was well suited to
linkage analysis, we tested the possible involvement of the
LDLR gene. Two polymorphic markers were studied: D19S394 and
D19S221, which lie 250-kilobase telomeric and 1-megabase centromeric,
respectively, to the LDLR gene on chromosome 19.24 Using
the MLINK program, the highest 2-point lod scores were obtained for
marker D19S394 at 2.28 and 2.14 (
=0, complete and incomplete
penetrance, respectively), therefore establishing linkage between 1
allele of the LDLR gene and
hypercholesterolemia in the family (Figure
II). Sequencing each exon of the proband LDLR gene led to the
identification of a 664 proline to leucine defective mutation
(LDLR-P664L) and its detection in all 6
hypercholesterolemic-linked members of the family. This
recurrent mutation has already been reported. It results in a receptor
with a reduced binding affinity for LDL and in delayed processing of
the precursor form of the protein in cultured cells (2B+3 functional
classes).32 Informative marker (5' TGn and
3'HVR) analysis showed no cosegregation of a
particular APOB haplotype with
hypercholesterolemia, excluding implication of
an APOB mutation different from R3500Q and R3531C (data not shown).
Statistical analysis revealed significant differences
(P<0.001) in total and LDL-cholesterol between
carriers of the P664L mutation (mean value and 95% confidence interval
for total cholesterol and LDL-cholesterol were
8.68 [8.05 to 9.32] and 6.20 [5.64 to 6.76] mmol/L,
respectively) and family members lacking the mutation (mean value and
95% confidence interval for total cholesterol and
LDL-cholesterol were 5.49 [5.06 to 5.92] and 3.60 [3.17
to 4.05] mmol/L, respectively). Mean total
cholesterol expressed as MoM for age and gender was 1.67
for the 2 subjects carrying only the LDLR-P664L mutation and 1.12 for
the group without the 2 mutations screened (Figure
III). On the other hand, an effect of the
isolated APOB-R3531C mutation on plasma cholesterol levels
was not detectable. Subjects carrying only the APOB-R3531C mutation (6
subjects) showed lower mean lipid levels (5.22 mmol/L or 1.06 MoM
for total cholesterol and 3.27 mmol/L for
LDL-cholesterol) than the 10 subjects without the 2
mutations screened (5.65 mmol/L or 1.12 MoM and 3.81 mmol/L,
respectively) (Figure III). Furthermore, we did not detect
significant interaction between the 2 mutations on total and
LDL-cholesterol levels. Using raw total
cholesterol concentration values, the interaction test was
barely significant (P=0.05), but using these data expressed
as MoM for age and gender, the test was no longer significant
(P=0.09) (Figure III). However, thorough
analysis of our data indicates that carriers of the 2 mutations
all have higher total cholesterol levels than LDLR-P664L
heterozygotes (Table II) and that the mean adjusted total
cholesterol of the first group of patients is higher than
that of the second group (Figure III).
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There is no doubt that the R3531C mutation causes reduced binding of LDL to the LDL receptor in vitro.9 16 17 However, our results additionally support that this reduction is not sufficient to cause hypercholesterolemia in vivo in heterozygotes. The evidence that the R3531C mutation alone does not cause hypercholesterolemia stems from functional, epidemiological, and linkage analysis.
First, R3531C functional consequences measured in vitro are only half of the reduction observed with the R3500Q mutation. In the total of 12 R3531C heterozygous subjects who have now been reported, the overall binding affinity of LDL particles compared with reference was 57%,20 whereas it was 34% for the R3500Q mutation.9 LDL particles from individuals with the R3531C mutation were 74% as effective at promoting growth as normal LDL in a U937 cell assay,20 whereas the relative growth rates using LDL particles from heterozygous R3500Q and R3500W subjects were about 50% that of normal LDL particles.13 Ligand-defective Cys3531 LDL particles accumulated and comprised a mean of 58% of the total particles present, whereas the mass ratio of Gln3500 to Arg3500 LDL was 73:27.20 Defective apoB Cys3531 LDL itself has been calculated to have 27% of normal affinity compared with <10% for Gln3500 particles.9 LDLR mutations and the R3500Q mutation in APOB each lead to impaired LDL receptor and apo B-100 interaction, but FDB-R3500Q patients tend to have a milder phenotype (significantly lower plasma total and LDL-cholesterol and subsequent lower incidence of clinical atherosclerosis).10 11 12 Because the R3531C mutation in vitro causes only 50% of the reduction for the R3500Q mutation, in vivo consequences are expected to be reduced or undetectable.
Second, APOB-R3531C proband screening suffered from ascertainment
bias attributable to patient status. Sixteen of 27 probands reported
(Table I) were identified among either patients with
hyperlipidemia who were attending lipid
clinics9 18 19 20 or patients with CAD6 16 19
among whom patients with hypercholesterolemia
are naturally overrepresented. Therefore, the presence of
hyperlipidemia could be a consequence of the study
design and not attributable to any mutation. Despite this bias, only 15
of 27 probands heterozygous for the R3531C mutation displayed
hypercholesterolemia.6 9 16 18 19 20
For example, of the 4 CAD patients identified by Ludwig et
al,16 only 2 had
hypercholesterolemia and 1 had the rare E4/4
apolipoprotein E genotype that is associated with increased
plasma cholesterol. Recently, Pullinger et
al20 reported no statistically significant difference in
lipids between 24 affected and 18 unaffected individuals from 9
kindreds. This difference became significant only when all published
data were combined. However, the possible coexistence of another
mutation, as in the proband we report, and evident ascertainment bias
could explain how the level of significance was reached. This
hypothesis is in agreement with data published by Tybjaerg-Hansen et
al6 reporting the first 7 R3531C probands identified in
the general population. This was a very large study (9255 women and
men) without ascertainment bias attributable to patient status. None of
the R3531C probands identified in the general population had plasma
total or LDL-cholesterol elevation, suggesting that this
mutation is not sufficient to cause high cholesterol
levels, ie, FDB. Tybjaerg-Hansen et al6 also clearly
showed that the frequency of the R3531C mutation was not increased in
patients with hypercholesterolemia or
ischemic heart disease compared with the general population. In
contrast, the frequencies of the R3500Q mutation identified in exactly
the same populations were clearly increased.6 Although the
frequencies of these 2 APOB mutations are the same in the general
population, only APOB-R3500Q causes sufficient
hypercholesterolemia to be a risk factor for
ischemic heart disease. Furthermore, all studies except
116 that have examined the presence of either of these
mutations in the same patient population consistently showed a
lower frequency of R3531C versus R3500Q in
hyperlipidemic or CAD
patients.6 9 18 19
Finally, in support of this theory is the fact that there was an absence of simple cosegregation of this mutation with hypercholesterolemia in 11 of 12 families studied.9 18 19 20 The description of our informative French family, the largest studied so far, shows no cosegregation of the R3531C mutation with hypercholesterolemia that is linked to a defective LDLR mutation. Contrary to our investigation, most of the family studies did not definitely exclude the involvement of an LDLR gene defect18 20 nor the implication of the third locus (FH3) associated with autosomal dominant hypercholesterolemia.1 33 Of the 8 R3531C probands reported by Tybjaerg-Hansen et al,6 1 had ischemic heart disease and hypercholesterolemia, but a family history of these traits was absent.
However, when associated with the LDLR-P664L mutation, the APOB-R3531C
mutation seems to enhance hypercholesterolemia.
Heterozygote carriers of the APOB-R3531C mutation alone have mean
adjusted total cholesterol levels lower than subjects
without the 2 mutations screened, whereas carriers of the APOB-R3531C
mutation and the LDLR-P664L mutation have mean adjusted total
cholesterol levels higher than the carriers of the
LDLR-P664L mutation alone (Figure III). This inversion of
influence, although not statistically significant, suggests that the
APOB-R3531C mutation enhances the hypercholesterolemic
effect of the LDLR-P664L defect. This is characteristic of a
susceptibility mutation and fits well with the in vitro effects of this
mutation.
In conclusion, the APOB-R3531C substitution, in view of its in vitro effects and our family study, is possibly a susceptibility mutation that, when present with other factors (genetic or environmental), slightly increases cholesterolemia. However, it is not sufficient in itself to cause hypercholesterolemia and should not be considered as an allelic variant leading to FDB.
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Acknowledgments |
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This work was funded by grants from Université Paris V, Inserm-Progres, Arcol, and Parke-Davis. We wish to thank Dr Pascale Benlian for help in identifying the LDLR mutation and Dr David Marsh for careful reading of the manuscript and valuable comments.
Received May 16, 2000; accepted May 29, 2000.
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