© 1995 American Heart Association, Inc.
Articles |
Differences in the Phenotypic Characteristics of Subjects With Familial Defective Apolipoprotein B-100 and Familial Hypercholesterolemia
From the Departments of Research and Internal Medicine, University Hospital, Basel, Switzerland.
Correspondence to A.R. Miserez, MD, Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas TX 75235-7200.
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Abstract |
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Abstract Familial defective apolipoprotein B-100 (FDB) is a recently identified autosomal-dominantly inherited disorder caused by a point mutation in the apolipoprotein (apo) B gene. To determine whether the phenotypic characteristics in FDB subjects are similar to those in subjects with familial hypercholesterolemia (FH), 76 kindreds fulfilling the clinical criteria for heterozygous FH/FDB were characterized using molecular biological techniques. Allele-specific polymerase chain reaction (PCR) at the apoB locus was used for diagnosis or exclusion of FDB. PCR-based methods for detection of two point mutations (V408M and P664L) at the LDL receptor (LDLR) locus, cosegregation analysis using eight restriction fragment length polymorphisms (RFLPs) at the LDLR locus, or the exclusion of FDB confirmed the clinical diagnosis of FH. Three kindreds were not included because of a missing cosegregation between a particular haplotype and the FH phenotype. We predicted that a similar number of kindreds would be detected in the two groups, assuming comparable prevalences of the diseases in our population and similar phenotypic characteristics. However, only nine kindreds were identified with the FDB mutation compared with 64 kindreds with FH (P<.0001). From these 73 kindreds, 28 FDB heterozygotes and 129 FH heterozygotes were compared using multivariate analysis. There were no differences between these two groups with respect to age, sex, and apoE genotype distribution, lipoprotein(a) concentrations, body mass index, blood pressure, and smoking habits. However, FDB subjects demonstrated significantly lower concentrations of total cholesterol (8.1 versus 10.2 mmol/L, P<.001), LDL cholesterol (6.3 versus 8.2 mmol/L, P<.001), and triglycerides (1.3 versus 1.8 mmol/L, P=.025) and higher concentrations of HDL cholesterol (1.4 versus 1.2 mmol/L, P=.015) than subjects with FH. In contrast to FH, female FDB subjects tended to have higher concentrations of total cholesterol (8.9 versus 7.5 mmol/L, P=.032) and LDL cholesterol (7.1 versus 5.7 mmol/L, P=.026) than FDB males. The same results regarding total and LDL cholesterol and sex differences were observed when individual data of 238 FDB and 415 FH subjects from the literature were compared. In addition, FDB subjects showed much larger total cholesterol fluctuations than FH subjects (median of intraindividual coefficients of variation: FDB, 14.5%; FH, 5.3%; P<.001). In summary, these results demonstrate that FDB subjects tend to have a milder form of hyperlipoproteinemia than FH subjects and that only a part of the subjects with FDB fulfill the established criteria for identifying FH.
Key Words: apolipoprotein B-100 • LDL receptor • mutations • genotype • phenotype
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Introduction |
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LDL particles are catabolized primarily via the LDLR pathway, mediated by the interaction between the apoB molecules, the apolipoprotein moieties of the LDL particles, and the hepatic LDLRs. ApoB molecules are synthesized in hepatocytes and secreted in VLDL, where they function as ligands for the LDLR after conversion of VLDL to LDL. Binding of apoB to the LDLR results in cellular internalization of the LDL particles and their lysosomal degradation.1 Hence, on one hand, defects of the receptor as observed in FH or, on the other hand, defects of the ligand as observed in FDB result in increased LDLC concentrations.1 2 Defective binding of LDL particles to their receptors due to structural defects of apoB was recently identified in patients with moderately increased LDLC concentrations.3 4 5 This metabolic disorder was designated as FDB. A markedly reduced (20% to 32% of normal) binding of LDL particles isolated from subjects with FDB to LDLRs of cultured fibroblasts and a subsequent decrease of the clearance of LDL in turnover studies have been demonstrated in this disorder.5 6 FDB is caused by a single base substitution (G to A) at nucleotide 10 7087 in exon 26 of the apoB gene, creating an arginine-to-glutamine substitution at the codon for amino acid 3500.8 A high prevalence of FDB was found in the United States and in several European countries, in particular north of the Alps (prevalence in Switzerland, 1/210), suggesting that the origin of this point mutation is in central Europe.9 In a review published in 1990, the average TC concentration of the 41 FDB subjects described was considerably lower than that reported in the literature for subjects with FH.2 However, other reports investigating the clinical features of FDB in comparison to FH did not provide evidence for differences in the phenotypic characteristics between these two disorders.10 11 12 Thus, FDB and FH have been regarded as clinically indistinguishable.11 If this conclusion is correct and if the prevalences of the two disorders are similar in a given population, then the number of kindreds with FDB and the number with FH in a sample of kindreds presenting clinically with FH (FH/FDB) would be expected to be similar. To test this hypothesis, families clinically characterized as FH/FDB were identified and studied for the presence of defects in the apoB and the LDLR gene. Unexpectedly, the frequency of kindreds with FDB was much lower in our sample than that of kindreds with FH. Further analyses presented in this article confirmed our suggestion that differences between the phenotypic characteristics of FDB and FH subjects exist.
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Methods |
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Subjects
The analysis of individuals with FDB or FH in this study was based on a sample of 76 unrelated white kindreds from the German- and French-speaking parts of Switzerland north of the Alps. Most of the families were of autochthonous Swiss origin; no families from the Italian-speaking part (south of the Alps, approximately 290 000 inhabitants) of Switzerland were included. In a first step, kindreds were identified by lipid specialists from our lipid clinic and from other Swiss university hospitals according to the classic criteria for primary hypercholesterolemia: a family history of hypercholesterolemia (type IIa hyperlipoproteinemia: fasting pretreatment LDLC exceeding the 95th percentile for the Swiss population corrected for age and sex,13 14 normal fasting TG) present in three or more members from at least two generations and inherited in an autosomal-dominant fashion; a family history of premature atherosclerosis; and family members with tendon xanthomas, xanthelasmas, or arcus lipoides corneae. All kindreds were screened for FDB. For confirmation of FH, all FDB-negative kindreds were screened for two point mutations at the LDLR locus. Except in two kindreds with a LDLR point mutation, cosegregation analysis using RFLPs at the LDLR locus was performed. Kindreds with a missing cosegregation between a particular haplotype and the FH phenotype were excluded. In a second step, we included all individuals from the remaining families who had been characterized as affected by FDB (confirmed by allele-specific PCR) or FH (confirmed by PCR-based methods for direct mutation detection, by cosegregation analysis, or by the exclusion of the FDB mutation) or as not affected by both disorders (control subjects). Exclusion criteria for the individuals were secondary causes of hypercholesterolemia (diabetes mellitus, hypothyroidism, kidney or liver diseases) or missing data. To verify the results from our sample of FDB and FH subjects, 37 articles were retrieved from MEDLINE, and individual data for 653 subjects characterized as affected either by FDB or by a particular LDLR defect were collected and compiled in a database.
Lipoprotein Analyses
LDLC was determined from overnight fasting blood samples after
precipitation with heparin, and HDLC was determined after precipitation
with phosphotungstic acid and magnesium ions. Lipid concentrations were
measured using enzymatic colorimetric methods
(Boehringer Mannheim). Pretreatment values only (no
lipid-lowering diet or drugs) were used for further
analyses.
DNA Analyses
Total genomic DNA extraction from white blood cells was
performed as described previously.9 A combined
allele-specific and asymmetric PCR-based method was used for
screening for the apoB defect at nucleotide 10 708 causing
FDB.15 PCR amplification and subsequent digestion with
Nla III or Pst I, respectively, were performed
for screening for the presence of the V408M and P664L mutations in the
LDLR gene.16 17 Eight RFLPs (assessed by Southern blots or
by PCR amplification and subsequent digestion) at the restriction sites
of Taq I, Stu I, Hinc II,
Ava II, ApaLI (5'), Pvu II,
Nco I, and ApaLI (3') were used for cosegregation
analysis at the LDLR locus as described
elsewhere.18 The RFLPs extended from intron 4 to the
noncoding region, 14.6 kb downstream to the 3' end of exon 18 of the
LDLR gene and spanning a total distance of approximately 43.3 kb.
Restriction isotyping of human apoE was performed by gene amplification
and cleavage with Cfo I (isoschizomere of Hha
I).19
Statistical Methods
Interactions of different variables were studied with MANOVA
using the SUPERANOVA program. Independent
parameters in MANOVA were age; sex; pretreatment
concentrations of TC, LDLC, HDLC, VLDLC, and TG; lipoprotein(a); apoE
genotypes; body mass index; diastolic and systolic
blood pressure; smoking; the presence of xanthomas, xanthelasmas, or
arcus lipoides; coronary artery disease (assessed by
coronary angiography or defined by the presence of myocardial
infarctions); cerebrovascular disease (defined by the presence of
strokes); and peripheral vascular disease (assessed by
angiography or defined by the presence of intermittent claudication).
Differences of means within the three groups (FH, FDB, and control) of
normally distributed values (TC, LDLC, HDLC, VLDLC, body mass index,
and blood pressure) were assessed with ANOVA and Scheffé's F
tests and differences of not normally distributed values (age, TG,
lipoprotein[a]) by the Kruskal-Wallis test. Relative frequencies of
clinical features (sex; apoE genotypes; frequencies of obesity,
hypertension, smoking, xanthomas, xanthelasmas, and arcus lipoides; and
coronary artery, cerebrovascular, and peripheral
vascular disease) within the three groups were assessed by
2 tests for homogeneity (Brandt-Snedecor).
Significant differences between the groups stratified by sex were
assessed by unpaired t tests for normally distributed
values, by U tests (Mann-Whitney) for not normally
distributed values, and by Fisher's exact tests for frequencies.
Regression analysis was performed to assess age dependency of
the different variables. Univariate analyses
and regression analyses were studied using the
STATVIEW II program. Normally distributed data are
presented as mean±SD, not normally distributed data as median
(range), and frequencies as percentages (ratios of absolute numbers).
The probability P to observe zFDB or
less kindreds with FDB from a sample of n kindreds
(n=zFDB+zFH
where zFDB is the number of kindreds with FDB,
and zFH is the number of kindreds with FH;
fFDB,obs=zFDB/n is
the observed frequency of kindreds with FDB, and
fFH,obs=zFH/n is the observed
frequency with FH) was calculated using
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Results |
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From the 76 kindreds, there were nine kindreds with the G-to-A substitution at the apoB locus (FDB) and 64 kindreds with LDLR defects (FH). In these 64 kindreds, two with a mutation in exon 9 (V408M, FH-Afrikaner 2; three FH subjects included) and two kindreds with a mutation in exon 14, in the growth factor repeat C of the receptor (P664L, FH-Gujerat; 6 FH subjects), were detected. In 50 kindreds (110 FH subjects), FH was confirmed by cosegregation analysis, including two kindreds (five FH subjects) that were confirmed by direct detection of the point mutation as well. In 12 kindreds, FH was confirmed by exclusion of FDB (15 FH subjects). Three kindreds were not included because of a missing cosegregation of a particular haplotype with the FH phenotype. The observed frequency of kindreds with FDB in the sample was 9/73 or approximately 0.12, with a 99% confidence interval of 0.05 to 0.27 (or 4/73 to 19/73). The expected frequency based on the assumed prevalences of FDB and FH in Switzerland was 51/73 or approximately 0.70. The probability value to find a frequency of nine or fewer kindreds with FDB in the sample (fFDB,obs
0.12 or 9/73 with fFDB,exp=0.70)
was P<.0001. A total of 263 individuals (comprising 28 heterozygous FDB subjects, 129 heterozygous FH subjects, and 106 unaffected family members serving as control subjects) were included.
No differences were observed between FH, FDB, and control groups with
respect to age, sex, apoE genotype distribution, body mass
index, systolic and diastolic blood pressure, or smoking
habits (Table 1
). Differences in lipoprotein(a)
concentrations in a subset of FDB and FH subjects were not
statistically significant.
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Differences between FDB and FH groups were detected in our sample
(Table 2) for mean TC (Fig 1), LDLC,
HDLC, VLDLC, TG concentrations, and the TC-HDLC ratio but not for the
relative frequencies of tendon xanthomas, xanthelasmas, arcus lipoides,
and coronary artery, cerebrovascular, and
peripheral artery disease. Differences between males and
females regarding mean TC and LDLC concentrations were detected in FDB
but not in FH. In all three groups, there was a significant
age-dependent increase in TC and LDLC but not in HDLC
concentrations (Table 2). In contrast to FH, there was an
age-dependent increase in the TC-HDLC ratio in FDB subjects. In
contrast to FH and control, there was no age-dependent increase in
VLDLC concentrations in FDB subjects. In FH, TG concentrations were
significantly higher than in FDB and control groups, and HDLC
concentrations were significantly lower than in FDB and control (Table 2). Serial measurements of TC levels in FDB subjects revealed striking
fluctuations (up to 40%), in particular in young FDB subjects aged
between 15 and 40 years. The median of intraindividual coefficients of
variation (CV) was higher in FDB than in FH subjects (FDB: n=14,
CV=14.5%; FH: n=23, CV=5.3%; P=.0005; Fig 2).
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indicates FDB subjects >30 years; 
,
subjects 
, FH subjects >30 years; and 
, FH
subjects
In the compiled data from the literature including 238 FDB and 415 FH
subjects, the differences between FDB and FH regarding mean TC and LDLC
concentrations were confirmed (TC, P.0047; LDLC,
P.0078), except in FH-Gujerat (TC, P=.0213;
LDLC, P=.13; Table 3). The differences
between males and females regarding mean TC (P=.0001) and
LDLC (P=.0004) concentrations in FDB but not in FH were also
confirmed by the compiled data (Table 3). The significant
age-dependent increases in TC and LDLC but not in HDLC
concentrations were detected in the compiled data as well: in the FDB
sample and in the FH samples of 26 or more subjects (n
26,
P.0079; Table 3).
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The frequency of xanthomas in the compiled data from the literature was
significantly lower in FDB than in FH when the samples of FH subjects
for comparison were large enough (n44, P.0107; Table 3
)
but not in our sample of only 28 FDB subjects (Table 2;
P=.09). There was a predominance of xanthomas in females
with FDB, which was significant in the compiled data from the
literature including 65 FDB subjects (P=.0249) but not in
our sample of only 28 FDB subjects (Table 2; P=.07). In FH
subjects from our sample, there was a predominance of coronary
artery disease in males (n=129, P=.007) that was not found
in FDB subjects from our sample (n=28, P=.24) or by
analyzing a larger number of FDB subjects from the compiled data
characterized for the presence or absence of coronary artery
disease (n=79, disease present in FDB males: 47.5%, present in
FDB females: 51.3%; P=.74).
In addition to these differences between FDB and FH, two further
associations were detected. In FH subjects with xanthomas, there was an
absolute but also relative predominance of a homozygosity for the apoE
3 allele (E3/3; P=.003) compared with the
heterozygous apoE genotypes (E2/3, E2/4, and E3/4). In FDB
subjects suffering from strokes, there was a predominance of the apoE
4 allele (E2/4, E3/4; P=.025).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study revealed quantitative differences between FDB and FH with respect to mean pretreatment lipoprotein concentrations and differences with respect to the modulation of the phenotype by independent factors. There are three lines of evidence for quantitative differences between the two disorders. First, significantly lower mean TC and LDLC concentrations in FDB heterozygotes compared with FH heterozygotes were detected in our sample (Table 2
). Second, significantly lower mean TC and LDLC
concentrations in FDB heterozygotes compared with FH heterozygotes also
were detected in a compilation of 238 FDB and 415 FH subjects from the
literature, even when FDB was individually compared with each of the
LDLR mutations (Table 3). Third, an unexpectedly (if one assumes no
differences between FDB and FH) low number of nine FDB kindreds was
found in a sample of 73 (FDB and FH) kindreds selected according to the
clinical characteristics of FH/FDB. The selection of the kindreds for
this study was based on the clinical criteria for familial forms of
hypercholesterolemia and was blinded with
respect to the differential diagnosis of FDB versus FH. The ratio of
the observed frequencies of the two disorders
(fFDB,obs/fFH,obs) in the sample should
reflect (after exclusion of kindreds not affected by either FDB or FH,
thus fFH,obs=1-fFDB,obs) the proportion of the
prevalences of the two disorders
(gFDB/gFH) in a given population if the
chance of selection were identical for kindreds with FDB and FH, ie,
the subjects from the FDB and FH kindreds would fulfill the same
clinical criteria. If we assume that FDB and FH cause exactly the same
phenotypic features and the prevalences of FDB and FH are the same in a
given population (gFDB=gFH), then the expected
frequencies of the two disorders (fFDB,exp and
fFH,exp) in the study sample should be identical also.
Furthermore, if FDB and FH cause exactly the same features but the
prevalence of FDB is approximately 1/210, as has been estimated for the
Swiss population,9 and the prevalence of FH is
approximately 1/500, as it is assumed to be for white populations
without a founder effect,1 then we would expect to find
even more kindreds with FDB than with FH in our study sample. However,
the observed frequency of FDB kindreds (fFDB,obs=0.12 or
9/73) was much lower than expected (fFDB,exp=0.70 or
51/73). On the other hand, the expected frequency fFDB,exp
was clearly above the upper 99% confidence limit of
fFDB,obs. Even if one assumes an equal prevalence for FDB
and FH (fFDB,exp=fFH,exp=0.50), this would
still be the case. Similar ratios of observed and expected frequencies
in samples of subjects clinically diagnosed as FH were also found in
other populations studied (Table 4). Hence, the
frequencies of FDB kindreds observed in our study sample and in all
these other countries were unexpectedly low. Since this discrepancy is
very unlikely to have occurred by chance alone (P<.0001),
the clinical criteria defined above appear to be much more appropriate
to identify kindreds with FH due to LDLR defects than those with FDB.
This indicates that affected subjects from FH families better fulfill
the clinical criteria than those from FDB families. Hence, from this
observation, there must also exist differences in the phenotypic
characteristics of these two disorders. An exception might be
FH-Gujerat (P664L) (see Reference 1111 and Table 3), where TC but not
LDLC concentrations were significantly higher in FH subjects. In our
data, there were only two of 64 families with this particular
mutation.
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Three kindreds in which the clinical characteristics of FH (at least
LDLC concentrations >95th percentile) did not cosegregate with a
particular LDLR haplotype were not included in our study. It is
possible that a missing cosegregation in a family could be due to a
normocholesterolemic LDLR mutation carrier.
However, the probability that a subject carrying an LDLR defect does
not fulfill our inclusion criteria because of an LDLC concentration
below the 95th percentile is small. Since LDLC concentrations are
normally distributed, this probability can be roughly estimated by
z=(r95GP-µFH)/SDFH.
The FH subjects in Table 3 were studied using molecular biological
techniques; thus, normocholesterolemic FH carriers
or FDB subjects would have been detected. Using the overall mean LDLC
concentration of these 242 FH subjects (µFH=7.8,
SDFH=±1.6 mmol/L) and an average 95th percentile from the
Lipid Research Clinics study data20
(r95GP=4.6 mmol/L), z is -2.0 and
thus P<.03. Therefore, a sample defined by our inclusion
criteria should represent more than 97% of the subjects
carrying an LDLR mutation in a population. In line with this expected
low frequency (3%), there was only one subject (in one of the excluded
families) who was normocholesterolemic but shared
with her hypercholesterolemic family members the
haplotype corresponding to the LDLR defect. The other two families have
been excluded because there was in each family a
hypercholesterolemic subject with a haplotype that
could not be detected in her or his mother or in any of the brothers or
sisters. Extensive studies using further DNA markers suggested
recombinational events in these latter two families. To rule out the
question of influence on the results of possible
normocholesterolemic LDLR mutation carriers, an
extended analysis was performed. However, the inclusion of all
the potentially (clinically and/or molecular genetically) affected
individuals from these three families (eight
hypercholesterolemic subjects, one
normocholesterolemic subject) did not change our
results.
In 1986, FDB was detected in subjects who had been investigated because of an only moderate hypercholesterolemia (TC, 6.5 to 7.8 mmol/L) and the lack of the classic clinical features of FH. Thus, this first report described the disorder as a cause for moderate hypercholesterolemia in comparison to FH.4 There are further findings that are in line with our observation of quantitative differences in the phenotypic characteristics between FDB and FH:
In FDB, a gene dosage effect, as it has been described in
FH,1 appears to be missing. In both subjects homozygous for
FDB reported so far, TC was only mildly increased6 21
compared with subjects homozygous for FH (TC in FH homozygotes, 16.8 to
25.9 mmol/L).22 23 In the case reported by März et
al,6 the TC and LDLC concentrations in the FDB homozygote
(mmol/L: TC, 8.6; LDLC, 6.9) were similar to those of the heterozygous
members of the same family (mmol/L: TC, 7.6 to 8.6; LDLC, 6.0 to 7.1;
in subjects of the same generation). In the case reported by Funke et
al,21 the TC concentrations in the FDB homozygote (7.7 to
8.6 mmol/L) were between those of her heterozygous father (9.2 to 11.1
mmol/L) and her heterozygous mother (6.5 mmol/L). In both FDB
homozygotes, TC and LDLC concentrations were similar to those in the
238 FDB heterozygotes whose data are compiled in Table 3. This is
clearly in contrast to FH, where LDLC concentrations are usually
sixfold to 10-fold increased in homozygotes compared with only a
twofold to threefold increase in heterozygotes.1 24
Furthermore, both FDB homozygotes described hitherto, a 54-year-old
man and a 31-year-old woman, did not show any evidence for
coronary or peripheral artery
atherosclerosis. This is also in clear contrast to FH
homozygotes, in whom coronary artery
atherosclerosis frequently has its clinical onset
before the age of 10 years. FH homozygotes usually succumb to the
complications of coronary artery disease before age 20
years.24 In addition, in both FDB homozygotes, treatment
with HMG-CoA reductase inhibitors caused a pronounced
decrease of TC and LDLC concentrations (as reported by März et
al,6 by 26% and 31%, respectively) in contrast to FH
homozygotes, who are usually resistant to HMG-CoA reductase
inhibitors.24 In FDB homozygotes, binding or
uptake of LDL is greatly diminished but not completely abolished. The
residual binding activity of LDL from an FDB homozygote was
approximately 20% of normal.6 The residual binding of the
LDL to the receptor has been demonstrated on the one hand to be due to
a receptor-mediated uptake of the LDL subfractions of the lowest
densities (1.019 to 1.034 kg/L) in this article by the remaining
amounts of apoE on these particles. On the other hand, in the LDL
subfractions of intermediate density (1.034 to 1.040 kg/L), the
defective apoB displayed some residual receptor binding by
itself.6 The apoB molecule is modeled as a belt of about
2.0x5.4 nm with an average length of about 58.5 nm, which surrounds
the LDL particle.25 Arginine 3500 is suggested to
stabilize two clusters of amino acids (3147 to 3157 and 3359 to 3367)
that have been assumed to ensure the binding of the apoB to the LDL
receptor.6 Since the radius of the LDL particle may vary
considerably and, according to this model, also that of the
apoB,25 the stabilization of the two amino acid clusters
may be of importance to maintain the binding affinity of the apoB to
the LDL receptor. In FDB, the two amino acid clusters may be disrupted
in LDL particles smaller than 9.6 nm and larger than 10.1 nm (LDL
subfractions of 1.040 to 1.063 kg/L and of 1.019 to 1.034 kg/L), as
suggested by experiments of März et al.6 After
removal of the apoE-containing particles, these subfractions showed no
receptor binding. On the other hand, LDL particles of 9.6 to 10.1 nm
(subfractions of intermediate density or 1.034 to 1.040 kg/L) might be
of the optimal size to allow a residual receptor-mediated binding
despite the loss of the stabilizing amino acid arginine at position
3500 or, in other words, despite the FDB mutation. LDL particles
smaller than 9.6 nm were the only subfractions in FDB homozygotes with
completely abolished binding and uptake.6 In contrast to
these results, defective LDL receptors of FH homozygotes often show a
total or near total inability to bind or take up LDL particles. In a
review by Goldstein and Brown22 of 57 FH homozygotes,
there were 31 subjects showing a functional LDLR activity below 2% of
normal.
Although differences between FDB and FH are quite obvious in homozygotes, they are much more difficult to detect in heterozygous subjects. A major problem is the contribution of the normal allele to the phenotypic characteristics of a heterozygote. Another reason could be that the expression of the hyperlipoproteinemia in heterozygotes may be modulated by environmental factors and also may be dependent on age. Both appear to be the case in FDB rather than in FH. Differences between FDB and FH may therefore be difficult to detect if only adult individuals are included in the analysis. This might be a reason why previous studies investigating the phenotypic features of FDB10 11 12 did not provide evidence for differences in phenotype between FDB and FH. Thus, a study design with inclusion of individuals from all age groups, exclusion of individuals for whom the clinical diagnosis was not consistent with the molecular biological methods, and use of multivariate analysis including other factors known to modulate the lipoprotein levels was performed.
In FDB, moderate expression or absence of hypercholesterolemia has been observed by several authors.2 9 12 21 26 27 Numerous FDB subjects with TC and LDLC concentrations within the normal range have been described.
In FH, variable expression has been described as well.22 23 However, FH is highly penetrant at all ages,22 and individuals with LDLR defects and LDLC concentrations below the 95th percentile of the general population are rare, as demonstrated above. In some of these cases, the missing elevation of LDLC has been suggested to be caused by an interaction with other genes. In a family including normocholesterolemic subjects with LDLR defects, a dominant gene suppressing the hypercholesterolemia has been demonstrated to be responsible for the absence of elevated levels in some of these individuals.28
In the present study, a large number of unrelated families was studied. Thus, a lipid-lowering or a lipid-increasing effect of another gene present in one of these families is not expected to bias our results, as could be the case if the same number of subjects had been collected from a few unrelated families only.
Besides quantitative differences between FDB and FH regarding mean lipoprotein concentrations, there was evidence for at least two further differences with respect to the modulation of the phenotype by independent factors.
First, in FDB, there were differences between males and females (higher
mean TC and LDLC concentrations in FDB females, Table 2). These
differences were confirmed by an independent sample of 238 FDB subjects
from the literature showing significant differences as well (Table 3).
The predominance of xanthomas in FDB females was significant in the
compiled data from the literature including 65 FDB subjects but not in
our sample of only 28 FDB subjects. Both sex-specific differences
were not detected in FH subjects from our sample (Table 2) or in FH
subjects from the compiled data.
A lipid disorder (familial dysbetalipoproteinemia) affecting apoE, the
other ligand known to bind to the LDLR, results in a markedly reduced
binding of apoE-containing VLDL remnants to the LDLR. This disorder is
characterized by lower TC and LDLC concentrations in women than in men,
onset of hyperlipidemia approximately 10 years later in
women, and a distinct influence of estrogens on
hyperlipoproteinemia.29 In
contrast to this disorder, sex-specific LDLC-lowering effects
appear to be absent in women with FDB. In FDB subjects of the
present study, TC and LDLC concentrations were significantly higher
in women than men, the onset of hyperlipidemia was not
different between women and men, and female FDB subjects showed the
highest TC and LDLC concentrations (Fig 1). The differences between the
two disorders, each characterized by functionally impaired binding
either of apoB or apoE, suggest a different role for the two
apolipoproteins in the sex-specific mechanisms modulating the LDLC
levels. Furthermore, because of higher LDLC concentrations, females
with FDB potentially lose their advantage with respect to
atherosclerosis after the age of 41 to 60 years (Fig 1). Thus, the predominance of coronary artery disease in males
with FH, which has been detected in our sample and in other
studies,24 is expected to be less distinct in FDB than in
FH. This conclusion is consistent with the compiled data of 79
subjects, which revealed no sex-specific difference in FDB with
respect to coronary artery disease.
The second difference between FDB and FH was the presence of marked spontaneous cholesterol fluctuations (including normal and hypercholesterolemic values) in the FDB subjects available for repeated measurements. The median of the intraindividual coefficient of variation in FDB subjects (14.5%) in the present study was larger than in subjects with FH (5.3%) but also larger than that in subjects with type III hyperlipoproteinemia (5.1%)30 or healthy subjects (4.9% to 7.1%).31 32 33 In contrast to these observations, in FH heterozygotes the elevated levels can often be diagnosed at birth and persist throughout life,22 a lipid-lowering diet usually reduces the TC concentrations only by 10% to 15%.24
The striking fluctuations and the distinct increase in early adulthood
(15 to 40 years, Figs 1 and 2) support the hypothesis that there are
compensatory mechanisms either reducing the production rate of
the apoB-containing particles or increasing the catabolism of
apoB-defective LDL particles (eg, by an increased uptake of
apoE-containing VLDL remnants and large LDL particles or by an improved
binding and uptake of LDL particles of intermediate
size).6 In heterozygotes, differences between FDB and FH
in lipoprotein concentrations may be caused by a decreased uptake of
apoE-containing particles by the defective LDLRs in FH
subjects.34 This hypothesis is supported by the
significantly higher TG and VLDLC concentrations in FH compared with
FDB subjects in our sample (Table 2). Increased TG concentrations have
been previously reported in approximately 10% of the subjects with
FH.1 In an animal model for FH (the WHHL rabbit),
differences between LDLR-defective animals and controls with respect to
LDLR-mediated uptake of apoE-containing particles have been
demonstrated.35 Hence, these observations suggest that
LDLR defects may be associated with an impaired clearance of
apoE-containing particles. On the other hand, on the assumption that
the clearance of apoE-containing TG-rich VLDL remnants and large LDL
particles is unaffected in FDB because both the LDLR and apoE are
intact, this mechanism would explain the lower TG and VLDLC
concentrations found in FDB when compared with FH. In our sample, the
TG and VLDLC concentrations in FDB subjects were not higher than those
in the control group (Table 2). These observations are
consistent with the finding that, in FDB, the uptake of
apoE-containing particles is unimpaired.6 Furthermore,
since VLDL remnants are the precursors of LDL, the apoE-mediated
binding and uptake of apoE-containing VLDL remnants may contribute to
the only moderate hyperlipoproteinemia found in
FDB compared with FH. In FDB heterozygotes, upregulation of hepatic
LDLRs may enhance the residual binding of large LDL particles and the
clearance of the apoE-containing VLDL remnants before they are
converted to LDL. In FDB heterozygotes, an upregulation of LDLRs
results in the production of 100% functional active LDLRs.
Thus, an upregulation is probably more effective with respect to the
apoE-mediated uptake than an upregulation of LDLRs in FH
heterozygotes to the same extent, which is expected to result in
production of only about 50% functional active LDLRs. This is
consistent with the observation in FDB subjects that an
upregulation of LDLRs by HMG-CoA reductase inhibitors
reduces the TC and LDLC concentration quite considerably, even in FDB
homozygotes.6 21
However, the spontaneous increase in TC concentrations of young FDB subjects suggests that such mechanisms may decompensate rapidly with increasing age and that a large percentage of individuals with the FDB mutation develop hypercholesterolemia. Regarding the marked fluctuations detected in FDB subjects, these compensatory mechanisms appear to be very susceptible to environmental factors. As long as the VLDL production is normal, and predominantly large LDL particles are produced that can be cleared by the LDL receptors, the hyperlipoproteinemia may remain moderate. Dietary change, such as to a carbohydrate-rich diet, results in the formation of large VLDL particles that are converted to smaller LDL particles.6 25 26 Since the size of the LDL particles has been demonstrated to be of importance for the binding of the defective apoB to the LDLR,6 dietary changes may therefore cause a change in the binding affinity of the apoB. In FH heterozygotes, however, the apoB is stabilized by arginine at position 3500; thus, smaller LDL particles generated under these conditions are still able to bind to the functional active LDLRs, and the LDLC concentrations in FH subjects are less susceptible to these factors.
In summary, the present study reveals several lines of evidence for differences in the phenotypic characteristics between subjects with FDB and FH. In FDB, mean pretreatment TC and LDLC concentrations were lower than in FH. In contrast to FH, there were higher TC and LDLC concentrations in FDB females than in FDB males. Significantly larger fluctuations in subjects with FDB than in subjects with FH suggest differences between FDB and FH in ability to compensate for metabolic changes (eg, those caused by environmental factors). However, once decompensated, heterozygous individuals with FDB may be hardly distinguishable from heterozygous individuals with FH. Thus, the inclusion of other (in particular, pediatric) members of the patient's family may be helpful in distinguishing between these two lipid disorders.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Postgraduate Course for Experimental Biology and Medicine of the University of Zürich and by the "Wissenschaftlicher Kredit" of the University Hospital of Basel, Switzerland. We are most grateful to numerous physicians in our country who provided excellent anamnestic data and helped us collect blood samples from FDB and FH subjects and their relatives. The technical help of N. Chiodetti is gratefully acknowledged.
Received October 14, 1994; accepted July 25, 1995.
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References |
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