Download Variable Expression of the Mutation in Familial Defective

973
Variable Expression of the Mutation in Familial
Defective Apolipoprotein B-100
J.J. Gallagher and N.B. Myant
Although most subjects with familial defective apolipoprotein B-100 (FDB) have raised plasma low-density
lipoprotein (LDL) levels, a few have LDL levels within the normal range. We have previously identified two
normocholesterolemic FDB heterozygotes in an affected family. Results obtained from a study of this family
are compatible with a major genetic contribution to the normocholesterolemia in the two heterozygotes.
However, our findings are not compatible with inheritance of a variant normal allele at the apolipoprotein
B locus in this family that neutralizes the effect of an FDB allele on the plasma LDL level. Polymorphic
variations at the apolipoprotein E and LDL receptor loci did not explain the presence of normal LDL levels
in the two heterozygous FDB subjects. (Arteriosclerosis and Thrombosis 1993;13:973-976)
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KEY WORDS •
expression
familial defective apolipoprotein B-100 •
F
amilial defective apolipoprotein (apo) B-100
(FDB) is a rare disorder of lipoprotein metabolism caused by a mutation in the apoB gene.1
The mutation gives rise to the substitution of arginine
for glutamine at residue 3500 in apoB-100, the protein
of low-density lipoprotein (LDL). The effect of the
Arg->Gln substitution is to reduce the binding of LDL
by the LDL receptor by more than 90%.2 In most FDB
heterozygotes, this gives rise to hypercholesterolemia
caused by the accumulation of LDL particles containing
mutant apoB-100.3 However, the plasma LDL concentrations in age-matched heterozygotes vary over a wide
range, and in some affected families the serum LDL
cholesterol (LDL-C) and total cholesterol levels are
normal in one or more heterozygous carriers of the
mutation.4-3 We have described a family with a total of
nine FDB heterozygotes, two of whom had normal LDL
levels on repeated measurement in the absence of
treatment/ and Friedl et al5 report a heterozygous man
whose plasma LDL-C level was 3.5 mmol/L. Friedl et
al5 suggest that the low LDL level in their FDB patient
was due to the presence of a mutation in the normal
("non-FDB") allele at the apoB locus that leads to
underrepresentation of its product in the plasma. In the
present work we tested the hypothesis that the normal
cholesterol levels in our two atypical FDB heterozygotes are also caused by the presence of a variant
normal allele at the apoB locus that counteracts the
hypercholesterolemic effect of the mutant allele. Our
results were not consistent with this hypothesis.
Methods
Subjects
Four FDB heterozygotes from an FDB family, together with three of their unaffected relatives, were
Received July 31, 1992; accepted February 4, 1993.
From the MRC Lipoprotein Team, Hammersmith Hospital,
London, UK.
Correspondence to MRC Lipoprotein Team, Hammersmith
Hospital, Ducane Road, London W12 OHS (Dr Myant).
plasma LDL level •
variable gene
examined. The characteristics of the seven subjects are
shown in Table 1. The members of the AE family shown
in Table 1 and the Figure are a branch of the more
extended family described in Reference 4. The upper
limit of the normal range for plasma LDL-C concentration in men and women was taken as 5.0 mmol/L6 and
3.3 mmol/L for children aged 2 through 14 years.7 FDB
was diagnosed by analysis of the subject's genomic
DNA, as described.8 High-affinity binding of LDL by
normal LDL receptors on fibroblasts in culture was
determined by a competitive binding assay, as previously
described.9 The test sample of LDL was added in
triplicate, at increasing concentrations, in the presence
of 125I-labeled normal LDL (3 /*g protein/mL medium),
to wells containing monolayers of fibroblasts after induction of receptors by growth in the presence of
lipoprotein-deficient serum for 48 hours. The concentration of test sample required to displace 50% of the
125
I-LDL from the fibroblasts (IQo) was calculated from
the competition curves, as described.9
Plasma Lipids and Lipoproteins
Plasma total cholesterol, high-density lipoprotein
cholesterol (HDL-C), and total trigryceride concentrations were determined as described elsewhere.4 Plasma
LDL-C concentration was estimated by using the
Friedewald equation.10
Haplotype Analysis
Genotypes were determined at five polymorphic sites
in the apoB gene and at two polymorphic sites in the
LDL receptor gene. For the apoB gene, genotypes at
the insertion/deletion site in exon 1, at the restriction
sites for Xba I and EcoRI, and at the 3' hyper variable
region (HVR) were determined as described.4 The sizes
of the HVR fragments were determined by comparison
with DNA markers of known lengths, kindfy supplied by
Alison Dunning. The genotype at the Apa\A site in the
apoB gerie was determined by the method of Young and
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Arteriosclerosis and Thrombosis
TABLE 1.
Subject
Vol 13, No 7 July 1993
Characteristics of AE Family FDB Heterozygotes and Their Relatives
FDB
1
2
1
2
n
in
Sex
Age
(y)
M
F
M
F
M
M
F
1
60
35
34
37
8
6
TC
TG
LDL-C
(mmol/L)
8.7
6.2
7.2
3.8
5.0
3.6
3.6
0.8
1.1
1.0
0.8
0.9
0.5
0.9
7.3
4.3*
5.6t
2.6
3.4t
2.7t
2.6
ApoE
phenotype
BMI
3/3
3/3
3/3
3/3
3/3
3/3
3/3
23.1
20.3
23.4
23.6
223
18.7
18.7
FDB, familial defective apolipoprotein (apo) B-100; TC, plasma total cholesterol; TG, plasma triglyceride
concentration; LDL-C, low-density lipoprotein cholesterol; BMI, body mass index. Subject 11 is III 5 in Reference 4.
"Mean of two determinations separated by 3 years. All lipid measurements were made before treatment with
lipid-lowering drugs. For pedigree see Figure.
tMean of three or more determinations made at intervals of at least 6 months,
t i normal plasma LDL-C concentration.
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Hubl.11 Genotypes at the Avail and Hindi polymorphic
sites in the LDL receptor gene were determined as
described.4 Haplotypes at the apoB and LDL receptor
loci were deducible from the genotypes in all seven
individuals.
AE family
a/x
B/C
Q
A/B
I
Pedigree showing the haplotypes of the normal and mutant
alleles at the apolipoprotein (apo) B locus and the low-density
lipoprotein (LDL) receptor alleles in the AE family. Half-filled
squares are heterozygous familial defective apoB-100 males
and open circles are normal females. ApoB haplotypes a, b, c,
d, and x are shown in Table 2. Receptor gene haplotypes are
A, Ava//+ /Hinc//+; fl, Ava//+/Hinc//-; Q Ava/r/Hinc// + .
+
, Restriction site present; ', restriction site absent; *, normal
plasma LDL cholesterol concentration.
Results
ApoB Haplotypes
Five different apoB haplotypes, based on six variable
sites at or near the apoB locus, were identified in the
seven members of the AE family (Table 2). These
included the haplotype (designated x in Reference 4
and Table 2 and A in Reference 5) that specifies the
apoB allele bearing the mutation at codon 3500 in most
white FDB heterozygotes reported from Europe and
North America.4-5-12-13
The AE Family
The branch of the AE family investigated in this study
comprised seven individuals and included two heterozygous brothers (II 1 and II 3), one hypercholesterolemic and the other normocholesterolemic, and a heterozygous boy (III 1) who was normocholesterolemic.
The affected father of the two brothers (I 1) was
hypercholesterolemic. Their unaffected mother was
normocholesterolemic, but her age-adjusted LDL-C
level was close to the 90th percentile for women.14 The
two brothers were of similar age and body mass index,
both had apoE phenotype 3/3, and both were nonsmokers. The plasma LDL-C and total cholesterol levels in
the 8-year-old heterozygous boy were similar to those of
his normal 6-year-old sister.
The inheritance of haplotypes at the apoB and receptor
loci is shown in the Figure. The two FDB brothers (II 1
and II 3) inherited different normal (non-FDB) apoB
alleles from their mother. The two brothers also inherited
different LDL receptor alleles from their mother, II 1
receiving B and II3 receiving A. HI 1 inherited one of the
two receptor alleles with haplotype B present in his father.
Since his father was hypercholesterolemic, neither of
these alleles is likely to have had a significant cholesterollowering effect. From his mother, III 1 inherited a receptor allele with haplotype C.
Receptor binding of LDLs from II 1 and II 3 was
defective, and the defect was equally marked in the two
samples. The IC50 for LDL from II 1 was 12.2 ji,g/mL
and from II 3 was 13.9 /ig/mL compared with a mean
value of 3.4±0.24 ug/mL obtained from LDLs from
seven normal donors.
Discussion
Variation between different individuals in the expression of a harmful mutation may be explained by differ-
Gallagher and Myant
TABLE 2.
Variable Expression in Familial Defective ApoB-100
975
Haplotypes Bated on Six Variable Sites in or Near the ApoB Locus in a Family With FDB
Variable site
Haplotype
I/D
ApaU
X
a
b
c
d
E
Xbal
3500
Gin
Arg
Arg
Arg
Arg
Arg
EcoRl
HVR
49
47
41
29
43
35
Apo, apolipoprotein; FDB, familial defective apoB-100; I/D, insertion or deletion in exon 1; HVR, 3'
hypervariable region (number of 15-base repeats); +, insertion or restriction site present or ( - ) absent.
x is the haplotype in which the 3500 mutation in the apoB gene is present. 4 E is the haplotype of the
normal apoB gene in the normocholesterolemic FDB heterozygote referred to in Reference 5.
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ences in environmental and genetic factors that influence
the phenotype. In heterozygous familial hypercholesterolemia (FH), eg, interindividual variation in plasma LDL
levels and in clinical signs is probably due to a combination
of environmental effects, such as habitual diet, and genetic
effects, which may include differences in the nature of the
mutation in the mutant LDL receptor allele.'5 Hobbs et
al16 described an FH family in which some individuals
appear to have inherited a dominant gene that suppresses
hypercholesterolemia, resulting in normal plasma LDL
levels in FH heterozygotes who have inherited the suppressor gene.
In contrast to FH, all FDB heterozygotes have an
identical mutation in the mutant allele that gives rise to
their disorder. Apart from this difference from FH,
factors analogous to those responsible for variability in
expression of the FH gene are likely to be responsible
for variability in the plasma LDL concentration in FDB
heterozygotes. Nongenetic factors may well make a
major contribution to variability in the severity of
hypercholesterolemia in the FDB population as a
whole. However, a predominantly genetic basis for
variability seems more likely in cases in which one or
two FDB heterozygotes within a family are normocholesterolemic while other affected members are hypercholesterolemic. In the AE family, the two heterozygotes II1 and II 3 were both men in their mid-30s, both
ate a modified low-fat diet, and their body mass indexes
were similar. Nevertheless, II 1 was consistently hypercholesterolemic, whereas II 3 has remained normocholesterolemic in the absence of drug treatment throughout an almost 3-year period of observation. In addition,
the plasma LDL level in III 1, who had FDB, was similar
to that in his unaffected normocholesterolemic sister,
although both ate the same diet and had similar body
mass indexes. The absence of hypercholesterolemia in
this 8-year-old boy is unlikely to have been due to a
normally delayed expression of the FDB mutation, since
we have noted marked hypercholesterolemia in an FDB
heterozygote at age 2 years.4 These findings strongly
suggest a major genetic contribution both to the difference in plasma LDL levels in the sibling pair I I 1 and II
3 and to the similarity in the levels in III 1 and III 2.
In the FDB family described by Friedl et al,5 one of
six heterozygotes (J.H.) was normocholesterolemic.
J.H. had no siblings, so that a sibling-pair comparison
was not possible. However, the haplotype of his nonFDB allele at the apoB locus was different from that of
the non-FDB alleles in his five hypercholesterolemic
FDB relatives. Friedl et al5 suggested that J.H. had
inherited a rare mutant non-FDB allele at the apoB
locus, whose product was underrepresented in his
plasma. In support of this, they showed that the IQo of
J.H.'s LDL (determined by a competitive binding assay)
was two to three times the IQoS of the LDLs obtained
from his heterozygous relatives, and that this anomaly
was due to the presence of an abnormally high proportion of LDL particles in his plasma containing apoB
encoded by the FDB allele.
Taken by itself, our observation that AE I I 1 and II 3
inherited different non-FDB alleles from their unaffected mother is consistent with the possibility that the
apoB allele specified by haplotype c suppressed the
hypercholesterolemic effect of the FDB allele in II 3.
However, the presence of this allele in the AE family
would not explain the normal plasma LDL level in III 1,
who inherited from his mother a non-FDB allele (with
haplotype a) that was different from the non-FDB allele
in his heterozygous uncle (II 3). Moreover, the ICJOS of
LDL from II 1 and II 3 were increased to the same
extent. This is not compatible with the presence of an
abnormally high proportion of defective LDL particles
in the plasma of II 3, as observed in J.H. It should also
be noted that if the apoB allele with haplotype c were
responsible for the normal LDL level in II 3, the
presence of this allele in I 2 should have caused her to
have an abnormally low LDL level. In fact, her LDL
level was near the upper limit of the normal range.
These findings argue against the presence of a mutation
in the non-FDB allele at the apoB locus in the AE
family that leads to underrepresentation of its product
in the plasma.
The inheritance of maternal LDL receptor alleles by
the two brothers II 1 and II 3 is consistent with the
possibility that receptor allele A lowered the plasma
LDL level in II 3. However, if the presence of this allele
is a sufficient cause of the normal level in II 3, its
presence in I 2 should have caused her to be hypocholesterolemic. Subject III 1 inherited receptor allele B
from his hypercholesterolemic father and receptor allele C from his unaffected mother. Since his mother had
low plasma total cholesterol and LDL-C levels, it is
possible that the maternal receptor allele with haplotype C contributed to the low plasma LDL level in II 2
and III 1.
Although our findings are hard to reconcile with the
hypothesis that the normal LDL levels in two heterozygotes resulted from the inheritance of a variant normal
976
Arteriosclerosis and Thrombosis
Vol 13, No 7 July 1993
apoB allele, they do not exclude the possibility that the
normal levels in these individuals were caused by the
combined effects of several genes, each having a small
effect on the plasma LDL when acting alone. Possible
components of a polygenic system acting on the plasma
LDL include polymorphic variants at the apoB and
LDL receptor loci and at other gene loci that influence
the metabolism of LDL or the rate of production or
catabolism of its lipoprotein precursors. Variation at the
apoE locus cannot have contributed to variability in
plasma LDL level in this family because all subjects
investigated had the same apoE phenotype.
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Variable expression of the mutation in familial defective apolipoprotein B-100.
J J Gallagher and N B Myant
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Arterioscler Thromb Vasc Biol. 1993;13:973-976
doi: 10.1161/01.ATV.13.7.973
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