© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Clinical Expression of Familial Hypercholesterolemia in Clusters of Mutations of the LDL Receptor Gene That Cause a Receptor-Defective or Receptor-Negative Phenotype
From the Department of Internal Medicine (S.B., L.P., M.R., S.L.), University of Genoa; the National Institute of Health (A.C., I.B.), Rome; the Institute of Internal Medicine (M.A.), University of Palermo, Palermo; the Department of Internal Medicine (C.C., C.M.), University of Rome "Tor Vergata," Rome; the Department of Medical Sciences (S.M.), University of Padua, Padua; the Institute of Clinical Biochemistry (G.P.), University of Sassari, Sassari; the Department of Clinical and Experimental Medicine (A.P.), University of Naples "Federico II," Naples; the Institute of Medical Therapy (C.S.), University of Rome, Rome; the Department of Biomedical Sciences (M.G., S.C.), University of Modena and Reggio Emilia, Modena; and the Department of Pediatrics (I.R.), University of Turin, Turin, Italy.
Correspondence to Sebastiano Calandra, Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi, 287, 41100 Modena,-Italy. E-mail sebcal{at}unimo.it
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Abstract—Seventy-one mutations of the low density lipoprotein (LDL) receptor gene were identified in 282 unrelated Italian familial hypercholesterolemia (FH) heterozygotes. By extending genotype analysis to families of the index cases, we identified 12 mutation clusters and localized them in specific areas of Italy. To evaluate the impact of these mutations on the clinical expression of FH, the clusters were separated into 2 groups: receptor-defective and receptor-negative, according to the LDL receptor defect caused by each mutation. These 2 groups were comparable in terms of the patients’ age, sex distribution, body mass index, arterial hypertension, and smoking status. In receptor-negative subjects, LDL cholesterol was higher (+18%) and high density lipoprotein cholesterol lower (-5%) than the values found in receptor-defective subjects. The prevalence of tendon xanthomas and coronary artery disease (CAD) was 2-fold higher in receptor-negative subjects. In patients >30 years of age in both groups, the presence of CAD was related to age, arterial hypertension, previous smoking, and LDL cholesterol level. Independent contributors to CAD in the receptor-defective subjects were male sex, arterial hypertension, and LDL cholesterol level; in the receptor-negative subjects, the first 2 variables were strong predictors of CAD, whereas the LDL cholesterol level had a lower impact than in receptor-defective subjects. Overall, in receptor-negative subjects, the risk of CAD was 2.6-fold that of receptor-defective subjects. Wide interindividual variability in LDL cholesterol levels was found in each cluster. Apolipoprotein E genotype analysis showed a lowering effect of the
2 allele and a
raising effect of the 4 allele on the LDL
cholesterol level in both groups; however, the
apolipoprotein E genotype accounted for only 4% of the
variation in LDL cholesterol. Haplotype analysis
showed that all families of the major clusters shared the same
intragenic haplotype cosegregating with the mutation, thus suggesting
the presence of common ancestors.
Key Words: LDL receptor gene mutations • familial hypercholesterolemia • mutation clusters • receptor phenotype and clinical expression
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Introduction |
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Familial hypercholesterolemia (FH) is a common autosomal, codominant disease caused by mutations in the LDL receptor (LDL-R) gene. It is characterized by elevated plasma LDL levels, tendon xanthomas, premature coronary artery disease (CAD), and a family history of 1 or more of these.1 In most populations, FH has proved to be extremely heterogeneous at the DNA level: >600 mutations of the LDL-R gene have been reported so far1 2 3 (also see http://www.ucl.ac.uk/fh). Studies conducted in the rare patients with homozygous FH have shown that different types of mutations result in residual functional LDL-R activity that varies from <2% to 30%.2 4 This mutational heterogeneity explains, to some extent, the phenotypic variation found in FH homozygotes, in whom there is a strong correlation between residual receptor activity in cultured cells and the severity of the disease.1 2 4 A similar genotype-phenotype correlation is less clear in FH heterozygotes (with documented mutations in the LDL-R gene), because there is only a weak correlation between maximally induced LDL-R activity in cultured cells and the plasma LDL cholesterol (LDL-C) concentration. In these patients, clinical expression of FH is highly variable in terms of the severity of hypercholesterolemia and the age of onset and severity of CAD, as well as the response to diet and lipid-lowering therapy.5 A few studies on the genotype-phenotype correlation in heterozygous FH have been carried out in isolated populations (such as the French-Canadian), wherein a founder effect had caused the accumulation of many patients with a limited number of mutant alleles of the LDL-R gene.6 Although these studies have provided some clue about the effect of LDL-R gene mutations on the clinical expression of FH, their results should be treated with caution, as it is likely that such patients might have other genes and environmental factors in common that might contribute to their phenotype.
The marked heterogeneity of mutations of the LDL-R gene causing FH, which is present in most populations, makes it difficult to collect a sufficiently large number of patients with the same mutation and to compare the FH phenotype in groups of patients (of the same population) carrying different mutations at the LDL-R locus. Recently, Graham et al7 have investigated genotype-phenotype correlations in heterozygous FH carrying different mutations of the LDL-R gene. They showed that frameshift (FS) mutations were associated with higher levels of LDL-C than those found in missense mutations.
In an attempt to determine to what extent the type of genetic defect is an important determinant of FH phenotype, we have investigated the clinical expression of the disease in several groups (clusters) of FH heterozygotes with defined mutations of the LDL-R gene that have been identified in Italy over the last few years.
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Methods |
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Patients
The study included a group of 725 unrelated patients (index cases) with the clinical diagnosis of heterozygous FH. Four hundred ninety-five subjects had a diagnosis of "definite FH" according to the following criteria: (1) untreated LDL-C levels >95th percentile of the distribution in the Italian population, stratified for sex and age; (2) vertical transmission of hypercholesterolemia and a bimodal distribution of plasma LDL-C levels in the proband’s family; and (3) the presence of subjects with the FH homozygous phenotype in the family (n=72), tendon xanthomatosis in the proband or in at least 1 first-degree relative (n=215), or severe hypercholesterolemia in some prepuberal children of the proband’s family (n=208). Two hundred thirty subjects were classified as "probable FH," since either no xanthomas were detected in the probands or in their family members or no sufficient family data were available (Table 1
). Data on smoking habits,
arterial hypertension, diabetes, or other associated
diseases with relevant impact on cardiovascular risk
were collected.
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FH patients were considered to have coronary artery disease (CAD+) if they (1) had history of a documented myocardial infarction, coronary artery bypass graft, or percutaneous transluminal coronary angioplasty; (2) suffered from angina pectoris with a positive exercise ECG and thallium test or an abnormal angiogram; and (3) had silent ischemia that was detected during bicycle exercise testing and was confirmed by thallium test, stress echocardiography, or angiography. FH subjects were considered free of CAD (CAD-) if they had a negative bicycle exercise test. CAD occurring before 60 years of age was considered premature CAD. The families belonging to the FH clusters (see Results) were considered positive for the presence of tendon xanthomas and/or premature CAD if at least 1 adult member of the family was affected.
Informed consent was obtained from all subjects investigated, and, in the case of children, from their parents. The study protocol was approved by the human investigation committee of each participating institution.
Biochemical Analysis
Fasting plasma lipid concentrations were measured before any
hypolipidemic drug treatment. Total cholesterol,
triglycerides, and HDL-C levels were measured enzymatically
with commercial kits (Boehringer Mannheim GmbH) and an
automated analyzer (Hitachi model 704); LDL-C was calculated by
the Friedewald formula.
Assay of LDL-R Activity in Skin Fibroblasts
The assay of 125I-labeled LDL binding,
internalization, and degradation in cultured skin fibroblasts was
performed as described by Goldstein et al.8 Residual LDL-R
activity (calculated as the maximum rate of saturable
125I-LDL degradation) was expressed as a
percentage of the value obtained in control fibroblasts.
Analysis of the LDL-R Gene
Genomic DNA was extracted from peripheral blood
leukocytes by a standard procedure.9 All DNA samples were
digested by using 5 to 10 U/µg of several restriction enzymes,
separated by agarose gel electrophoresis, transferred to nylon
membranes, and hybridized with various LDL-R cDNA
probes.10 Polymerase chain reaction (PCR) amplifications
of the promoter region and exons 1 to 18 of the LDL-R gene were carried
out by using the primers reported by Hobbs et al2 in a
total volume of 50 µL containing 100 ng of genomic DNA, 10
mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 100 µmol of each of the 4
nucleotides, 10 pmol of each primer, and 2 U of
Taq DNA polymerase. Single-strand conformation
polymorphism analysis was performed by using a vertical gel
unit; 5 µL of PCR product was mixed with an equal amount of 95%
formamide, 20 mmol/L EDTA, 0.1% bromophenol blue, and 0.1%
xylene cyanole; denatured at 96°C for 5 minutes; snap-cooled in 4°C
ice water; and then loaded onto an 8% polyacrylamide gel
containing 5% glycerol. Electrophoresis was performed at room
temperature in a standard Tris-borate-EDTA buffer, pH 8.0, at 150 V for
2 hours. After electrophoresis, the gels were stained with silver. The
samples showing an abnormal single-strand conformation polymorphism
pattern were sequenced by using an automated fluorescence ABI
Prism 310 genetic analyzer (Applied Biosystems Inc) and
following the manufacturer’s recommendations. Mutations identified by
the automated sequencer were confirmed by manual
sequencing4 with the use of appropriate primers.
Several mutations reported in Table 2, 2A have been characterized
previously in our laboratory.4 10 11 12 13 14 15 16 17 18 19 20 21 22 23
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The screening of some mutations in the probands’ family
members was performed by using a variety of procedures: (1) Southern
blotting (deletion of exons 2 to 12 and exons 13 to
14)12 16 ; (2) PCR amplification of fragments of the LDL-R
gene with appropriate canonic primers, followed by digestion with
restriction enzymes (MspI for D200G, NlaIII for
V502M, HinfI for D558Y, and PstI for P664L); (3)
PCR amplification of fragments of the LDL-R gene with appropriate
mismatched primers, followed by digestion with restriction enzymes
(NdeI for 313+1 g
a, StyI for E207K,
RsaI for FS453, and StuI for G571E); (4) PCR
amplification of a specific allele (PASA) for
G528D20 ; and (5) heteroduplex analysis for
FS572.23 Details of the procedures adopted for screening
these mutations will be reported elsewhere.
Geographic Origin of Mutations
The putative geographic origin of each LDL-R gene mutation was
empirically established on the basis that individuals carrying a
specific mutation were proved to have stemmed from ancestors who had
been living in the same geographic area for at least 3 generations.
Haplotype Analysis of the LDL-R Locus
The intragenic haplotypes cosegregating with some mutant
alleles (D200G, FS453, and G528D) were constructed by using
diallelic markers present in the LDL-R locus. Genotyping was
performed by (1) Southern blotting (BstEII,
5'ApaLI, PvuII, NcoI, and
3'ApaLI); (2) restriction enzyme digestion of PCR-amplified
intron 7 (SmaI) and exons 8, 12, 13, and 15
(StuI, HincII, AvaII, and
5'MspI); and (3) single-strand conformation
polymorphism analysis (1413G/A in exon
10).24 25 26 27 28
The polymorphic tetranucleotide microsatellite D19S394 that is 250 kb telomeric to the LDL-R gene was genotyped by using fluorescently tagged, PCR-generated fragments29 that were electrophoresed in an Applied Biosystems automated DNA sequencer 373 A Leon and sized by Genescan 672 software (Applied Biosystems).
Screening for Familial Defective Apo B-100
All probands were screened for the presence of the R3500Q
mutation in the apolipoprotein (apo) B gene causing familial defective
apo B-100.30
Apo E Genotyping
The apo E genotype was determined by PCR amplification
of genomic DNA according to the procedure of Hixson and
Vernier.31
Statistical Analysis
Statistical analysis was performed by using the
SPSS 9.0 (SPSS Inc) program. Statistically significant
differences between groups or among groups for continuous variables
were evaluated by Student’s t test for unpaired data or
ANOVA, respectively. Triglyceride values, which were not
normally distributed, were logarithmically transformed before
analysis. Multiple comparisons among pairs of means based on
unequal sample sizes were performed by the Dunnett C method. Lipid
values were adjusted for sex, age, and body mass index (BMI) by linear
multiple regression analysis. When subjects were grouped by apo
E genotypes, their lipid values were also adjusted separately
in the receptor-defective group and the receptor-negative group for the
effect of each LDL-R gene mutation (ie, the lipid values for each
mutation were adjusted to the grand mean of the whole group).
Differences in the distributions of categorical variables were
assessed by the 
2 test with Yates’ continuity
correction when necessary. Genotype distribution for the apo E
polymorphism was determined by gene counting.
2 analysis was used to
test for Hardy-Weinberg equilibrium and to compare the observed
genotype distributions and allele numbers in CAD+ and
CAD - subjects. The proportion of the total phenotypic
variability of adjusted LDL-C and HDL-C values attributable to the apo
E gene polymorphism was calculated according to Boerwinkle et
al.32 The independent contribution of each
variable to CAD was evaluated by logistic regression
analysis, taking into account only those variables that
were found to be predictive in univariate analyses.
The independent categorical variables were coded as follows: 1 for
male and 0 for female sex; 0 and 1 for the absence and presence,
respectively, of arterial hypertension; 0, 1, 2, and 3 for
nonsmoker, former smoker, current light smoker (<10 cigarettes per
day), and current heavy smoker (>10 cigarettes per day), respectively;
0 for mutations causing a receptor-defective phenotype and 1
for mutations causing a receptor-negative phenotype; 1 for
genotypes 22 and 23; 2 for genotype
33; and 3 for genotypes 24, 34, and 44.
Age and lipid values entered the analysis as continuous
variables. Likelihood ratio statistics were used to compare models
with different combinations of predictors with an inclusion
significance level of 0.05 and an exclusion significance level of 0.1.
Odds ratios and 95% confidence intervals are also reported.
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Results |
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Mutations of the LDL-R Gene in FH Patients
Table 1
shows the number of families (unrelated index
cases) and subjects with the clinical diagnosis of "definite" or
"probable" FH, in whom an LDL-R gene mutation was detected during a
national survey that is still in progress. At this time,
analysis of the LDL-R gene mutation has been completed in all
families of patients with homozygous FH, in 42% of families with
definite FH (presence of tendon xanthomas or
hypercholesterolemic children), and in 15% of families
with probable FH. By extending the genetic analysis to the
relatives of index cases, we have been able to collect 
1000 FH
heterozygotes with identified mutations in the LDL-R gene.
Table 2 shows the mutations of the LDL-R gene found in our
index patients to date, the number of apparently "unrelated"
families carrying the same mutation, and the number of subjects
(heterozygotes, homozygotes, and compound heterozygotes) belonging to
these families. The allele designation indicates the putative
geographic origin of each mutation in our country. The table includes
mutations that (1) were discovered in Italian FH patients and were
reported previously by our group (see References 4 and
10 23 and the web site
http://www.ucl.ac.uk/fh); (2) were discovered in other countries and
reported in the literature but also found to be present in Italy
(see the web site); and (3) are reported for the first time in this
article (new). During this survey, we discovered that some mutations
are overrepresented in specific geographic districts of the
country. For example, D200G (FH Padua-1), though detected in all
districts of northern Italy, is found mostly in the Veneto region and
has so far not been detected in central and southern Italy. On the
other hand, G528D (FH Palermo-1) is found in southern Italy and Sicily
but not in northern Italy (Figure 1).
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The identification of several apparently unrelated individuals with the
same mutation allowed us to define the presence of clusters of
mutations and to compare the FH phenotype among these clusters.
In the present study, we arbitrarily defined a "cluster" as any
mutation group that included 5 or more apparently unrelated families.
At present, we have identified 12 such clusters (Table 3).
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Haplotype Analysis
Haplotype analysis was carried out in the largest clusters
(D200G and FS453 found in northern Italy and G528D found in southern
Italy and Sicily). In each cluster, the haplotype of intragenic markers
cosegregating with the mutation was the same in all families, thus
suggesting a common ancestor. D200G and FS453 cosegregated with the
following haplotypes: SmaI (-), StuI (+),
1413G/A (G), HincII (-), BstEII (-),
AvaII (+), MspI 5' (+), ApaLI 5' (+),
PvuII (-), NcoI (+), and ApaLI 3'
(+). G528D cosegregated with the following haplotype: StuI
(+), HincII (+), BstEII (-), AvaII
(-), ApaLI 5' (-), PvuII (-), NcoI
(-), and ApaLI 3' (-).Analysis of the
microsatellite D19S394, located 250 kb upstream from the LDL-R locus,
showed that the FS453 mutation cosegregated in all families with a
single allele (251 bp); the D200G mutation cosegregated with 3
alleles (227, 235, and 239 bp with a frequency of 0.18, 0.41, and
0.41, respectively); and the G528D mutation cosegregated with 4
alleles (243, 255, 259, and 263 bp with a frequency of 0.07, 0.41,
0.25, and 0.27, respectively). In 256 normal chromosomes, the
allele frequencies were as follows: 227 (0.05), 235 (0.10), 239
(0.05), 243 (0.09), 251 (0.10), 255 (0.12), 259 (0.12), and 263
(0.10).
FH Phenotype in Receptor-Defective and
Receptor-Negative Clusters
Table 3 shows the main clinical features of the
clusters. Lipid values reported in the tables were adjusted for age,
sex, and BMI. We separated the clusters into 2 groups designated
"receptor-defective" and "receptor-negative," respectively, on
the basis of (1) the residual LDL-R activity found by us and/or others
in cultured cells (fibroblasts) of homozygous subjects (>5% for
receptor-defective, <5% for receptor-negative)1 2 4 or
heterozygous subjects (>55% for receptor-defective, <55% for
receptor-negative) carrying the same mutation and (2) the presence of a
mutation leading to a frameshift and a truncated receptor (null
allele). One of the striking features emerging from the
analysis of these clusters is the large variability of plasma
LDL-C levels observed in each cluster, regardless of the type of
mutation of the LDL-R gene. The mean difference between the lowest and
the highest LDL-C values was 5.04±1.61 mmol/L in the
receptor-defective group and 6.13±1.58 mmol/L in the
receptor-negative group. Table 3 also shows that in the
receptor-negative group, the prevalence of tendon xanthomatosis in the
families (defined as the number of families in which at least 1 living
member had tendon xanthomatosis) and the prevalence of premature CAD
(defined as the number of families in which premature CAD had been
detected in at least 1 living member or had been reported as the cause
of death in at least 1 deceased family member) were respectively, 2.7-
and 2.1-fold those found in the receptor-defective group.
Table 4 shows the comparison of the
clinical features and lipid values between index cases (1 index case
for each unrelated family included in the major clusters shown in Table 3) and between all heterozygous subjects. Index cases of the
2 groups were comparable in terms of sex distribution, mean age, BMI,
smoking habits, and arterial hypertension. The unadjusted
mean plasma levels of total cholesterol and LDL-C were
significantly higher in the receptor-negative subjects, whereas plasma
triglyceride and HDL-C levels were similar in the 2 groups.
When the data were adjusted for age, sex, and BMI, plasma HDL-C levels
were found to be slightly but significantly lower in the
receptor-negative subjects. The percentage of index patients with
tendon xanthomas and CAD was higher in the receptor-negative
subjects.
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Considering all heterozygous subjects belonging to the clusters, the 2 groups were comparable in terms of mean age, sex distribution, arterial hypertension, and percentage of current and former smokers. BMI was found to be slightly higher in the receptor-defective group. Diabetic patients (1 patient in the receptor-defective group and 2 patients in the receptor-negative group) were not included.
The differences in the unadjusted and adjusted total
cholesterol, LDL-C, and HDL-C levels observed in the index
patients were clearly confirmed when we compared all subjects belonging
to the 2 groups (Table 4). In receptor-negative subjects, the
adjusted LDL-C and HDL-C levels were 18% higher and 5% lower,
respectively, than those found in receptor-defective subjects. Figure
2 shows the age-related increase of LDL-C level in both
groups; at each age interval, the mean LDL-C value was higher in the
receptor-negative subjects. The differences in the prevalence of tendon
xanthomas and CAD in the 2 groups were similar to those found in index
cases (Table 4).
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In both receptor-defective and receptor-negative subjects, the
genotype distributions of the apo E polymorphism were in
Hardy-Weinberg equilibrium (receptor-defective: 23 n=18, 24
n=2, 33 n=137, 34 n-31, and 44 n=1;
receptor-negative: 22 n=1, 23 n=24, 24 n=1, 33
n=230, 34 n=40, and 44 n=2), and no significant differences
in allele frequencies were observed between the 2 groups
(receptor-defective: 2 0.053, 3 0.854, and 4 0.093;
receptor-negative: 2 0.045, 3 0.879, and 4 0.076). In both
groups, plasma lipid values adjusted for sex, age, BMI, and the effect
of each LDL-R gene mutation were found to be influenced by the apo E
polymorphism. In comparison with subjects carrying the 33
genotype, in the receptor-defective group the 23
genotype was associated with lower HDL-C levels (1.09±0.24
versus 1.32±0.30 mmol/L, P<0.05) and with a tendency
to higher triglyceride levels (1.75±0.93 versus
1.39±0.65 mmol/L), whereas in the receptor-negative group,
23 was associated with lower LDL-C levels (6.99±1.18 versus
7.60±1.50 mmol/L, P<0.05). In both groups, subjects
carrying the 4 allele (24, 34, or 44
genotypes) had higher LDL-C levels than did subjects with other
genotypes (receptor-defective: 7.13±1.72 versus
6.49±1.36 mmol/L in the 33 group and 6.23±1.09 mmol/L
in the 23 group, P<0.05; receptor-negative:
8.25±1.33 versus 7.60±1.50 mmol/L in the 33 group and
6.99±1.18 mmol/L in the 22+23 group,
P<0.05).
CAD in Receptor-Defective and Receptor-Negative Clusters
Because it is well established that the prevalence of tendon
xanthomas and CAD is affected by age, we looked at the distributions of
these clinical features in subjects younger or older than 30 years of
age. In subjects 
30 years of age (71 in the receptor-defective group
and 112 in the receptor-negative group), no individual was detected
with CAD in either group, and only a few (2.8% in each group) were
found to have tendon xanthomas. When subjects >30 years were compared
(118 in the receptor-defective group and 186 in the receptor-negative
group), the prevalence of tendon xanthomas and CAD in receptor-negative
subjects was 2.4-fold (P<0.001) and 1.8-fold
(P=0.01), respectively, greater than that observed in
receptor-defective subjects.
Table 5 compares the clinical features
and lipid values in receptor-defective and receptor-negative subjects
>30 years of age who were CAD(+) or CAD(-). In both groups, age, the
prevalence of arterial hypertension, previous smoking
habits, and total cholesterol and LDL-C levels were higher
in CAD(+) patients. In the receptor-negative group, the male-to-female
ratio and BMI were significantly higher in CAD(+) patients, whereas in
the receptor-defective group, plasma triglycerides were
higher in CAD(+) patients. The prevalence of premature CAD (occurring
before 60 years of age in subjects >30) was significantly higher in
receptor-negative patients (23.2% versus 10.6%;
2=4.76, P=0.03),
with a crude odds ratio (95% confidence interval) of 2.55 (1.10 to
6.41).
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The CAD(+) distribution according to quartiles of age was examined in subjects >30 years of age. In the first and second quartile, the prevalence of CAD was similar in the receptor-defective and receptor-negative groups; in the fourth and especially the third quartile, the prevalence of CAD was much higher in the receptor-negative subjects (fourth quartile, 56.3% versus 33.3%, P=0.05; third quartile, 41.7% versus 16.1%, P=0.02).
Multiple logistic regression analysis, performed separately in the 2 groups in subjects >30 years of age, showed that independent contributors to CAD were (1) male sex (P=0.02), arterial hypertension (P=0.02), and LDL-C level (P=0.007) in the receptor-defective group and (2) male sex (P=0.001), age (P<0.0001), and arterial hypertension (P=0.02) in the receptor-negative group. This analysis also showed that in the receptor-defective group, age had a marginal effect on CAD (P=0.06), and in the receptor-negative group, the LDL-C level did not appear to be a good predictor for the development of CAD (P=0.08). In both groups, the apo E genotype did not significantly contribute to either premature CAD or total CAD.
Table 6 shows the results of multiple
logistic regression analysis performed in both groups taken
together to compare the risk of CAD associated with receptor-negative
versus receptor-defective mutations. This analysis showed that
in subjects >30 years of age with receptor-negative mutations, the
risk of CAD was 2.6-fold higher than that observed in subjects with
receptor-defective mutations.
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Mutation Spectrum and Clusters
In this study, we report partial characterization of the spectrum of mutations of the LDL-R gene discovered in Italy during a survey that started a few years ago and is still in progress. So far, our inventory consists of 71 mutations and includes 16 major rearrangements; 8 nonsense, 31 missense, and 11 frameshift mutations; and 5 splicing defects. During this study, we found that
4% of subjects with
clinically severe FH either do not carry detectable mutations in the
LDL-R gene (proximal promoter, coding sequence, and exon/intron
junctions) or show a phenotype that does not segregate with the
LDL-R gene haplotype within the family. This discovery is not
surprising, as other studies have reported that a certain proportion of
putative FH subjects do not have detectable mutations at the LDL-R
locus.33 34 35 From the mutation list shown in Table 2, the conclusion that emerges is that Italy is like the
United Kingdom,7 33 Germany,36 and
Japan,37 where many mutations have produced a highly
heterogeneous picture, in contrast to the situation found
in other countries (like Norway, Finland, and, to some extent,
Denmark), where few mutations account for a large proportion of FH
patients.38 39 40 For each index case, we tried to retrieve
as much family data as possible to define the geographic origin of the
ancestor likely to carry the mutation identified in the index case.
This family history allowed us to locate clusters of mutations in
specific areas of the country and to identify several clusters. To have
a sufficient number of subjects for the comparison of lipid values and
clinical features in patients carrying different mutations, we decided
to pool the clusters according to the predicted effect of each mutation
on LDL-R function (receptor-defective versus receptor-negative
clusters; Table 3). This analysis showed that even
in the setting of our large allelic heterogeneity,
heterozygous FH patients who carry receptor-negative mutations have a
more severe atherogenic lipid profile (higher LDL-C and lower HDL-C)
than do patients with receptor-defective mutations. These results agree
with previous observations made in population samples living in other
geographic settings. In French-Canadian FH heterozygotes carrying a
large deletion (>15 kb) in the promoter and the first exon of the
LDL-R gene (receptor-negative phenotype), the mean serum
cholesterol level was much higher than in heterozygous
patients with the W66G mutation (receptor-defective
phenotype).6 Afrikaner FH patients with a common
receptor-defective mutation (D206E) have significantly lower
cholesterol levels and milder disease than do Afrikaners
carrying a receptor-negative mutation (V408M).41 Vohl et
al42 compared FH heterozygotes carrying null alleles,
FH heterozygotes carrying defective alleles, and non-FH subjects.
They found that carriers of null alleles had the highest plasma
total cholesterol and LDL-C levels and the highest total
cholesterol–to–HDL-C ratio, whereas the carriers of
defective alleles had intermediate levels between null-allele
carriers and non-FH patients. Similar results, reported in other
studies,7 43 44 suggest that in FH heterozygotes (as
previously documented in FH homozygotes),1 4 6 the
difference in the magnitude of increase in plasma
cholesterol and LDL-C levels largely derives from
differences in the nature of the mutation in the LDL-R gene.
Prevalence of CAD
In index cases as well as in all heterozygous FH patients, the
prevalence of CAD in receptor-negative subjects (of both sexes) was
1.7-fold higher than that found in subjects carrying receptor-defective
mutations. Furthermore, when subjects >30 years old were further
stratified by age, we found that in receptor-negative patients, CAD
occurred at a younger age than in receptor-defective persons. This
observation is in keeping with that of Vohl et al,42 who
showed that in French-Canadian FH heterozygotes, the development of CAD
as estimated from the age at first coronary angiography or at
first revascularization occurred at an earlier age
in FH patients carrying a null mutation compared with carriers of
defective alleles. Recently, Gaudet et al44 compared the
expression of CAD assessed by coronary angiography in young (25
to 49 years) versus middle-aged (50 to 64 years) male FH heterozygotes
with receptor-negative mutations (>15-kb deletion, Y468X or R329X) and
with missense mutations (W66G, E207K, or C646Y); in this latter group,
92% of individuals carried the W66G mutation that produces a
receptor-defective phenotype. In both age groups, carriers of
receptor-negative mutations had higher plasma cholesterol
and LDL-C levels than did carriers of missense mutations, and their
relative odds of being affected by CAD before the age of 50 was
3-fold greater than that observed in patients with missense
mutations (in whom the receptor-defective phenotype was
prevalent).
Multiple logistic regression analysis performed in our subjects
who were >30 years of age showed that in both receptor-defective and
receptor-negative groups, independent contributors to CAD were male
sex, age, and arterial hypertension (Table 6). The
plasma level of LDL-C was a strong independent contributor to CAD in
the receptor-defective group but was a weaker contributor in the
receptor-negative group. This latter observation suggests that in our
receptor-negative subjects, any increase in LDL-C above certain
threshold level (eg, 8.0 mmol/L LDL-C) contributes less to the
development of CAD than do age, male sex, and arterial
hypertension. Taking into consideration all of these factors, the
multiple logistic regression analysis showed that in both sexes
>30 years of age, the risk of CAD is 2.6-fold higher in patients
carrying receptor-negative mutations.
In conclusion, compared with receptor-defective mutations, receptor-negative mutations are more strongly associated with premature CAD, this relationship being independent of common risk factors such as sex, age, and hypertension, as well as the other risk factors such as smoking habits, diabetes, and obesity,45 which were not contributors to CAD in our study.
Interindividual Variability of LDL-C Level
As previously reported,6 we found wide variations in
plasma lipid and lipoproteins, even in patients carrying the same
mutation. Table 3 shows that the wide interindividual
variation in LDL-C level persisted even after adjustment for sex, age,
and BMI and was present in all clusters regardless of the type of
mutation and its biological effect (receptor-negative and
receptor-defective). These variations may be due to genetic and/or
environmental factors that affect expression of the wild-type
allele (in the case of receptor-negative mutations) or both
alleles (in the case of receptor-defective mutations) or influence
other pathways of lipoprotein metabolism. Several groups
have investigated the role of common polymorphisms of several
candidate genes (such as apo E, apo B, and the LDL-R) that are known to
be associated with variations in plasma LDL, HDL, and
triglycerides in the general population. In light of the
point discussed above, the most reliable studies appear to be those
conducted on FH patients with identified mutations of the LDL-R gene.
Dallongeville et al46 were the first to show that LDL-C
levels were lower in the E3/2 subset than in the E3/3 or E4/3 subset of
heterozygous FH subjects with the >15-kb French-Canadian mutation.
These observations were expanded by Ferrieres et al,47 who
found that the contribution of the apo E polymorphism was different
between the sexes. Women with the 23 genotype had lower
total cholesterol, LDL-C, and apo B levels than did women
with the 33 genotype, whereas in men, the 22
genotype was associated with higher VLDL-C than was the
33 genotype. Overall, <20% of the variability for the
various lipid traits among these FH patients was explained by apo E
genotype. Surprisingly, these effects of apo E polymorphism
were not observed in a large group of FH heterozygotes carrying the
North Karelia mutation,48 although serum LDL-C levels were
found to be lower in subjects with the apo 42 genotype in
comparison with subjects with other genotypes.
Betard et al49 examined whether the phenotypic variation in lipoprotein and apo B levels observed in a sample of French-Canadians carrying the >15-kb deletion was associated with genetic variability of the "wild allele" (ie, nondeletion LDL-R gene). They found that some haplotypes, defined by using pairs of restriction fragment length polymorphisms of the LDL-R gene, contributed to quantitative variations in HDL-C and LDL-C levels in women but not in men. However, in FH subjects carrying the North Karelia mutation, Vuorio et al48 failed to find a significant association between PvuII and AvaII polymorphisms of the LDL-R gene or XbaI polymorphism of the apo B gene and plasma lipids and the prevalence of CAD.
In the present study, we found that in both receptor-negative and
receptor-defective groups, apo E genotype influences plasma
LDL-C levels. Compared with the 33 genotype, 22 and
23 genotypes have lower LDL-C levels, whereas the
opposite was true for genotypes containing the 4 allele.
Overall, apo E genotype accounted for 4.4% and 4.2% of the
variation in plasma LDL-C level (adjusted for sex, age, BMI, and the
effect of each LDL-R gene mutation) in receptor-defective and
receptor-negative groups, respectively. Therefore, apo E
polymorphism explains only part of the large interindividual
variability of plasma lipids and lipoproteins observed in each cluster
of our series, thus suggesting the presence of other genetic and
environmental factors. The important role played by environmental
factors has emerged from a recent study by Pimstone et
al,50 who compared lipid values and the prevalence of CAD
in FH Chinese subjects, with defined LDL-R gene mutations, living in
China and Canada. Higher LDL-C levels (and a higher prevalence of
tendon xanthomas and CAD), not accounted for by an effect of the LDL-R
gene mutation, were observed in FH heterozygotes living in Canada than
in those living in China. These differences could be ascribed, at least
in part, to the striking contrast in dietary fat consumption and level
of physical exercise between subjects living in Canada and those living
in China. It is reasonable to assume that these factors have a role to
play in our series, too. A systematic study of these factors in the
main clusters of our series is now in progress. Our preliminary dietary
survey, however, has failed to show striking differences in dietary
habits (in terms of total and saturated fat consumption) similar in
magnitude to that reported in the study of Pimstone et
al.50 A recent comparison (1994 to 1996) of dietary habits
between northern Italy (where, according to tradition, the diet is
presumed to contain more meat and dairy products) and southern
Italy (where a Mediterranean-type diet is presumed to be consumed) has
revealed that the differences in total and saturated fat intake are
fairly small (35% versus 33% and 13% versus 12%, respectively; A.
D’Amicis and A. Turrini, National Institute for Food and Nutrition,
personal communication, 2000). In this context, we need a very
large number of genotyped FH subjects to be able to ascertain
the contribution of diet to the interindividual variability of LDL-C
level.
Common Ancestor
The identification of clusters of families carrying the same
mutation in specific areas of the country raises the question of the
origin of these mutations (common ancestor versus recurrent mutations).
We examined the haplotype cosegregating with each mutation in the
largest clusters (D200G, FS453, and G528D). D200G has been reported in
other European countries in patients with different ethnic backgrounds,
suggesting the hypothesis that D200G is a recurrent mutation. However,
the observation that in our cluster, all patients share the same
intragenic haplotype and the mutation is mostly restricted to the
northeastern areas of Italy strongly suggests that D200G is identical
by descent in all patients of the cluster (which also includes 4
identified homozygotes). The G528D is a common mutation in southern
Italy and Sicily (the cluster includes 3 apparently unrelated
homozygotes and 2 related compound heterozygotes) but has been reported
in other European countries, too. Interestingly, G528D is fairly common
in Greece, where it accounts for 23% of unrelated FH heterozygotes and
shows 91% linkage disequilibrium with a single D19S394 microsatellite
allele (257 bp).51 In our series, all patients
carrying the G528D mutation share the same intragenic haplotype, but
the mutation was found to be linked with 4 different alleles of the
D19S394 microsatellite.
The finding that in our patients both D200G and G528D are in linkage disequilibrium with several alleles of the polymorphic microsatellite D19S394 telomeric to the LDL-R gene may be explained in several ways: (1) a de novo occurrence of these mutations in some probands; (2) a series of recombination events between the mutations in the LDL-R locus and the D19S394 locus; or (3) replication slippage of the D19S394 variable number of tandem repeats during meiosis. If we assume that recombinations between the 2 loci have occurred over time, we may estimate that numerous generations have elapsed since the common ancestor, given that there is an expected 1/400 chance of recombination between the D19S394 locus and LDL-R locus in a single generation.29 Apparently, the D200G and G528D mutations might date back to thousands of years ago. The FS453 mutation cosegregates in all 31 families not only with the same intragenic haplotype but also with a single allele of the D19S394 microsatellite (251 bp); in this case, we can assume that this mutation is considerably more recent, as it is limited to a restricted geographic area of the country.
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Acknowledgments |
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This work was supported by Telethon (project No. E. 0947), by a grant from the University of Genoa, and by a grant from the Italian National Institute of Health.
Received December 17, 1999; accepted March 17, 2000.
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