© 1999 American Heart Association, Inc.
Original Contributions |
Analysis of LDL Receptor Gene Mutations in Italian Patients With Homozygous Familial Hypercholesterolemia
From the Centro Prevenzione Arteriosclerosi (S.B., M.R.), Università di Genova, Genova; and the Dipartimento di Scienze Biomediche (S. Cassanelli, R.G., M.G., M.L.S., S. Calandra), Università di Modena, Modena, Italy.
Correspondence to Sebastiano Calandra, MD, Dipartimento di Scienze Biomediche, Università di Modena, Via Campi 287, I-41100 Modena, Italy. E-mail sebcal{at}unimo.it
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Abstract |
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Abstract—The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996. The age of the patients ranged from 1 to 64 years; one third of them were older than 30. Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L. The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r=-0.665; P<0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (
5% of
normal) and the highest plasma LDL cholesterol levels.
Twenty-nine patients (23 of whom were unrelated) were found to be
homozygotes at the LDL receptor locus. In this group we discovered 2
major rearrangements and 12 different point mutations (9 in the coding
region and 3 in splice sites). Some mutations (D200G, C358R, V502M,
G528D, and P664L) were found in 3 or more unrelated patients.
Patients with the same mutation shared the same haplotype at the LDL
receptor gene locus and came from the same geographic area. Ten
patients (9 of whom were unrelated) were found to be compound
heterozygotes. The mutations found in this group consisted of one large
deletion and 12 point mutations (11 in the coding sequence and one in a
splice site). In 3 compound heterozygotes we failed to identify the
second mutant allele at the LDL receptor locus. These observations
confirm the allelic heterogeneity underlying familial
hypercholesterolemia in the Italian population
and indicate that the variability of phenotypic expression of
homozygous familial hypercholesterolemia is, to
a large extent, related to the type of mutation of the LDL receptor
gene.
Key Words: homozygous familial hypercholesterolemia • LDL receptor gene • mutational analysis
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Introduction |
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Familial hypercholesterolemia (FH) is an autosomal codominant disorder characterized by an elevated level of plasma LDL cholesterol (LDL-CH), tendon xanthomas, and premature atherosclerosis. The genetic basis of FH is a lack of functional receptors for LDL on the cell surface.1 The heterozygous form of FH is estimated to be 0.2% in most populations.1 However in selected populations, such as Afrikaners of South Africa2 and French Canadians,3 the frequency of FH is much higher because of a founder effect. In European and North American populations homozygous FH is extremely rare, being estimated to occur in about 1 in 1 million births.1 Homozygous FH is characterized by a marked elevation of LDL-CH, cutaneous as well as tendon xanthomas, arcus cornealis, and generalized atherosclerosis developing during childhood. If untreated, death from myocardial infarction or sudden cardiac death is expected to occur by the end of the third decade of life.1
FH is heterogeneous at the molecular level; more than 200 mutations of the LDL receptor gene have been reported so far in FH patients.1 4 5 6 In some cases the effect of these mutations on LDL receptor function has been fully characterized.4 5 These receptor defects have been grouped into 5 major classes: (1) failure to express receptor protein synthesis; (2) defective transport of receptor precursors from the endoplasmic reticulum to the cell surface; (3) impairment of receptor binding to the ligand; (4) impaired internalization of the receptor–ligand complex because of defective clustering of LDL receptors into coated pits; and (5) defective receptor recycling.1 In many cases each functional class is associated with mutations in regions of the gene that encode one specific domain of the receptor protein.
During the past few years we have characterized more than 60 mutations of the LDL receptor gene in Italian FH patients.7 During this study we have had the opportunity to collect DNA samples, fibroblasts, and clinical data from FH homozygotes who have been referred to various Italian lipid clinics during the last decade and who were alive in 1994 or were born afterward. The aim of this study was the characterization of the mutations of the LDL receptor gene in these patients as the basis for future studies on the genotype–phenotype relationship, the response to pharmacological or nonpharmacological treatment, and the identification of suitable candidates for future gene therapy.
We performed DNA analysis in 39 patients, all of them Italians, with the clinical phenotype of homozygous FH. Molecular analysis has revealed that 29 of these patients were homozygotes and 10 were compound heterozygotes.
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Methods |
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FH Patients
In all cases the diagnosis of homozygous FH was based on the following criteria: (1) plasma or serum LDL-CH >10 mmol/L; (2) presence of tendon and cutaneous xanthomas at an early age; (3) an autosomal mode of inheritance of hypercholesterolemia in the kindred; and (4) the presence of primary hypercholesterolemia in the proband's parents. Detailed family studies were conducted specifically in 2 probands (compound heterozygotes 35 and 37 in Tables 3
and 4) who were found to have a relatively high
residual LDL receptor activity in fibroblasts and in whom we have been
able to identify only one mutant allele at the LDL receptor locus
(Table 4). In these cases plasma LDL-CH values found in the
probands' parents (8.5 mmo/L in the mother and 5.1 mmol/L in
the father of proband 35; 6.5 mmol/L in the mother and 6.6
mmol/L in the father of proband 37) suggested that the parents were FH
heterozygotes. This hypothesis was supported by the observation that
other subjects with primary
hypercholesterolemia were present in both
maternal and paternal lines of probands' families.
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Plasma lipids and lipoproteins were measured as previously reported.8 Informed consent was obtained from the patients or, in the case of children, from their parents. The study protocol was approved by the institutional human investigation committee of each participating institution.
Cardiological Evaluations
At the time of DNA analysis some patients had already
experienced a myocardial infarction or were affected by angina
pectoris. Most of the patients with overt coronary artery
disease (CAD) had undergone coronary angiography as well as
cardiac and carotid artery ultrasound examination. All
asymptomatic subjects had undergone
electrocardiography and, with the exception of
some children, ECG stress testing. Most patients have been under
treatment with LDL apheresis for several years, and some have also
received hypolipidemic drugs (HMG-CoA reductase inhibitors,
cholestyramine, and probucol). Several patients had undergone
coronary artery bypass graft surgery. Some patients died of
ischemic heart disease or sudden cardiac death during the study
(1994–1996).
Fibroblast Culture and LDL Receptor Activity
Skin biopsies were taken from 32 patients. The assay of
125I-labeled LDL binding, internalization, and
degradation by cultured skin fibroblasts was performed as described by
Goldstein et al.9 Residual LDL receptor activity
(calculated as the maximum rate of saturable
125I-LDL degradation) was expressed as a
percentage of the value obtained in control fibroblasts.
Southern Blot Analysis and Single-Strand Conformation
Polymorphism (SSCP)
Genomic DNA was extracted from peripheral blood
leukocytes or cultured fibroblasts by a standard
procedure.10 All DNA samples were digested using 5 to 10
U/µg of several restriction enzymes, separated by agarose gel
electrophoresis, transferred to nylon membranes, and hybridized with
various LDL receptor cDNA probes.8 SSCP was performed as
specified previously,11 using primers suggested in
previous reports.5 12 13 Polymerase chain reaction
(PCR)-amplified exons that showed an abnormal SSCP pattern were
sequenced directly using the ThermoSequenase sequencing kit
(Amersham).
PCR Amplification of Genomic DNA and Direct Sequencing
In one patient (No. 1), who was homozygous for a >15-kb
deletion eliminating the 3' end of the gene (downstream from exon 12),
exon 12 was amplified by PCR from genomic DNA using flanking
primers14 to ascertain whether this exon was involved in
the deletion. The incubation conditions were 94°C for 3 minutes, and
subsequently 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2
minutes for 35 cycles.
In the siblings 30 and 31 (carrying a deletion involving exon 13 to 15 region) we amplified the region spanning from exon 12 to intron 16 using primer 5' ACC GGA AGA CCA TCT TGG AGG ATG A 3' (as forward primer in exon 12) and primer SP85 (complementary to the 5' end of intron 16).12 In this PCR amplification we used high-fidelity Taq polymerase (Boehringer Mannheim) in the presence of 1.5 mmol/L MgCl2 under the following conditions: 94°C for 3 minutes, 94°C for 30 s, and 68°C for 7 minutes (10 cycles) and subsequently 94°C for 30 s and 68°C for 7 minutes (for the initial cycle), with a gradual increase in the elongation time of 20 s in each cycle, for 20 cycles. The PCR fragment corresponding to the deleted allele was purified by Jet-sorb (Genomed) and then sequenced using primer SP85.12
All patients were screened for the presence of the R3500Q mutation in apolipoprotein B by using a selective PCR amplification of the apo B gene.15
Northern Blot Analysis and Reverse Transcription-PCR
Amplification (RT-PCR)
Total cellular RNA was isolated by extraction in
guanidine-thiocyanate16 from cultured skin fibroblasts
that had been maintained in lipoprotein-deficient serum for 48 hours.
Northern blot analysis was performed as specified
previously.8
RNA (1 µg) from cultured fibroblasts of patients 27 and 28 and from a control subject was reverse-transcribed and amplified by PCR as specified previously.11 In the case of patient 27, RT-PCR was performed using the following primers: 5'-AAT GCA TCA CCC TGG AGA AAG TCT G-3' (6s; forward primer in exon 6) and 5'-GTT GTG GAA GAG GAC CAT ATC-3' (13as; reverse primer in exon 13). In the case of patient 28, RT-PCR was performed using the following primers: 5'-ATG ACA CCG TCA TCA GCA GGG A-3' (10s; forward primer in exon 10) and 5'-CTT GGT GAG ACA TTG TCA CTA TC-3' (15as; reverse primer in exon 15). The conditions were 95°C for 4 minutes and subsequently 95°C for 1 minute, 58°C for 1.30 minutes, and 72°C for 2 minutes, for 30 cycles. RT-PCR products were sequenced directly using the ThermoSequenase sequencing kit.
In 2 compound heterozygotes (No. 31 and 35), the search for the second mutant allele was performed on overlapping cDNA fragments (exons 1 to 9, 6 to 13, and 13 to 18) obtained by RT-PCR. The primers used for RT-PCR were the following: 5'-GCT GGA AAT TGC GCT GGA CCG TCG C-3' (1s; forward primer in exon 1) and 5'-CTG CAG ATC ATT CTC TGG GA-3' (9as; reverse primer in exon 9); primers 6s and 13as (see above); 5'-TTG TTG GCT GAA AAC CTA CTG TCC C-3' (13s; forward primer in exon 13); and 5'-CAA GGC CGG CGA GGT CTC AAG A-3' (18as; reverse primer in exon 18). For the amplification of exons 1 to 9 the conditions were 95°C for 3 minutes and subsequently 95°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes, for 30 cycles; for the amplification of exons 6 to 13 and exons 13 to 18, the conditions were 95°C for 3 minutes, and subsequently 95°C for 1 minute and 70°C for 1.30 minutes for 35 cycles. These fragments were sequenced directly using appropriate primers.
Haplotype Analysis
Haplotype analysis was performed in FH homozygotes
carrying the 5 most common mutations (D200G, C358R, V502M, G528D, and
P664L). Specific exons were amplified by PCR from genomic DNA using
previously reported primers12 13 and incubation
conditions.11 In the case of P664L mutation, because of an
abnormal NcoI restriction pattern detected by Southern
blotting, we also amplified intron 10 (using primers complementary to
intron 9 and intron 11)12 to check for the presence
of the NcoI restriction site in intron 10. This study
included DNA analysis of patients 23, 25, 26, and 38 as well as
that of 2 FH heterozygotes of Asian ancestry (carrying the P664L
mutation designated FH-Gujerat)17 18 and 2 British FH
heterozygotes with the same mutation.19 The DNA samples of
these patients were a kind gift from Dr A. Soutar (Royal Post-graduate
Medical School, London, UK).
Statistical Analysis
Values were given as the mean±standard deviation of the mean
(SD). Statistical comparison between groups was made using the
Student's t test.
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Results |
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Tables 1
and 3
show
some features of the patients with the clinical phenotype of
homozygous FH. The type and localization of the mutations in the LDL
receptor gene as well as the LDL receptor activity in skin fibroblasts
are listed in Tables 2 and 4. Our
series included 29 homozygotes (23 of whom were unrelated) (Tables 1 and 2) and 10 compound heterozygotes (9 of whom were
unrelated) (Tables 3 and 4). Plasma LDL-CH levels were markedly
increased (2.3- to 5.2-fold) above the 95th percentile value for the
population. There was no significant difference in the mean plasma
LDL-CH level between homozygotes (16.6±3.2 mmol/L) and compound
heterozygotes (17.4±4.3 mmol/L). In both groups the mean plasma
HDL cholesterol levels (0.80±0.22 in men and 0.81±0.27 in
women) were below the fifth percentile for our adult control population
(0.85 mmol/L for men and 1.02 mmol/L for women) (Tables 1 and 3).
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Severe cardiovascular involvement (as indicated
by the presence of 2- or 3-vessel CAD, myocardial infarction, or sudden
cardiac death) was observed in 16 of 25 patients older than 10 years.
These data are to be regarded as minimal figures, as we did not perform
advanced tests of ventricular function or structure,
especially in young patients.20 21 The residual LDL
receptor activity in cultured skin fibroblasts, measured in 32
patients, varied from <2% to 30% of the values found in fibroblasts
from control subjects (Tables 2 and 4). There was a
negative correlation between plasma LDL-CH level and the residual LDL
receptor activity (r=-0.655; P<0.003) (Figure 1). The highest levels of LDL-CH were
observed in 6 children (5 to 13 years of age) whose residual LDL
receptor activity was 5%.
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Mutations of LDL Receptor Gene in FH Homozygotes
The mutations found in FH homozygotes are listed in Table 2. In 2 patients (1 and 2) major rearrangements of the LDL
receptor gene were observed. The mutation found in patient 2 (a 5.5-kb
duplication involving exons 16 and 17) was previously reported in
detail by our group.8 In patient 1 the hybridization of
genomic DNA with a cDNA probe complementary to exons 1 to 11 (Ex 1–11
probe) resulted in normal fragments after DNA digestion with several
restriction enzymes, whereas no fragments were detected with a probe
complementary to exons 12 to 18 (Ex 12–18) (Figure 2). These findings suggested the presence
of a large deletion (>15 kb) eliminating the 3' half of the gene. An
additional Southern blotting of DNA digested with EcoRV
(whose restriction sites are located in exon 8 and at the 5' end of
intron 12) and hybridized with the Ex 1–11 probe, showed, besides the
expected 26-kb band, an abnormal band of approximately 20 kb, replacing
the expected fragment of 7 kb (Figure 2). This result suggested
that the EcoRV site in intron 12 was deleted. To ascertain
whether exon 12 was maintained we performed a PCR amplification of exon
12 using flanking primers. Because this amplification generated a
normal fragment (data not shown) we concluded that exon 12 was
maintained and the 5' boundary of the deletion was located at the 5'
end of intron 12. We were unable to define the 3' boundary of this
deletion. However, because a PCR amplification of genomic DNA, using
primers complementary to exon 12 and exon 18, was unsuccessful, we
assumed that all of exon 18 was deleted.
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In the other homozygotes we found 12 different point mutations, 9 in
the coding region and 3 in splicing sites (Table 2).
Mutations of LDL Receptor Gene in FH Compound
Heterozygotes
The mutations found in FH compound heterozygotes are listed in
Table 4. In patients 35, 37, and 39 we failed to identify one of
the 2 mutant alleles, despite an extensive analysis of the
proximal promoter, the coding sequence, and the exon–intron junctions
of the LDL receptor gene. In siblings 30 and 31 Southern blotting
revealed abnormal bands after genomic DNA digestion with several
restriction enzymes and hybridization with a cDNA probe complementary
to exons 12 to 18 (Figure 3). This
finding was consistent with the presence of an allele
carrying a 
5-kb deletion eliminating exons 13 to 15. The deletion of
exon 15 was also confirmed after PCR amplification of genomic DNA using
primers complementary to the 5' end of exon 12 and to intron 16.
Digestion of this PCR-amplified fragment (approximately 8 kb) with
EcoRV and HindIII demonstrated that the
EcoRV site in intron 12 and the HindIII site in
intron 15 were maintained. The presence of this mutation was also
confirmed by the analysis of LDL receptor mRNA in cultured
fibroblasts. RT-PCR amplification of the exon 12 to exon 18 region from
LDL receptor mRNA resulted in a fragment of 890 bp in the control and 3
fragments of 890, 424, and 346 bp in patient 31 (Figure 3). The
nucleotide sequence showed that (1) in the 890-bp fragment
exon 12 joined to exon 13 (indicating the presence of a nondeleted
allele); (2) in the 424-bp fragment exon 12 joined to exon 16
(indicating the presence of the deleted allele, as expected from
Southern blot results); and (3) in the 346-bp fragment exon 12 joined
to exon 17 (indicating the occurrence of an exon skipping in mRNA,
presumably transcribed from the deleted
allele).22
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Among the point mutations found in compound heterozygotes (Table 4), 11 were located in the coding sequence and one in a splice
site.
Haplotype Analysis
As shown in Table 2, some point mutations (D200G, C358R,
V502M, G528D, P664L) were observed in more than one apparently
unrelated FH homozygote. The haplotype analysis of the LDL
receptor gene showed that all patients with the same mutation shared
the same haplotype. The following polymorphic restriction sites
were examined: SmaI (intron 7); StuI (exon 8);
HincII (intron 12); BstEII (intron 12);
AvaII (exon13); MspI (exon 15); ApaLI
(intron 15); PvuII (intron 15); NcoI (exon 18);
and ApaLI (3'FLK). The haplotypes found were the following:
D200G (SmaI/-; StuI/+;
1HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+);C358R (SmaI/+; StuI/+;HincII/+; AvaII/-; MspI/+;ApaLI/-; PvuII/-; NcoI/-);V502M (SmaI/-; StuI/+;
HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+);G528D (StuI/+; HincII/+;BstEIi/-; AvaII/-; ApaLI/-;PvuII/-; NcoI/-; ApaLI/-); and
P664L (SmaI/-; StuI/+;HincII/-; AvaII/+; MspI/+;ApaLI/+; PvuII/-; NcoI/+). Furthermore, we discovered
that in patients 23, 25, and 26 the P664L mutation cosegregated with
the absence of an NcoI restriction site in intron 10 (data
not shown) that is normally found in our control subjects. The absence
of this site was also found in patient 38 (a compound heterozygote
carrying the P664L mutation). In sharp contrast, in 2 FH heterozygotes
of Asian descent (FH-Gujerat) and 2 British FH heterozygotes, the P664L
mutation was associated with the presence of the NcoI site
in intron 10. This suggests that the P664L substitution is a recurrent
mutation.
Effect of Splice-Site Mutations on LDL Receptor mRNA
The novel splice-site mutations found in patients 27 and 28 were
studied in detail with regard to the species of LDL receptor mRNA
generated by the mutant allele. In patient 27 (g
a+1 in intron
10) Northern blot analysis showed that the LDL receptor mRNA in
the proband's cells was apparently similar, in terms of size and
content, to that found in control cells (Figure 4). RT-PCR of the exon 6 to exon 13
region revealed the presence of a single 1109-bp fragment in the
control and 2 abnormal fragments of 1175 and 881 bp in patient 27. The
sequence of the 1175-bp fragment (corresponding to
mRNA1 in Table 5)
showed that exon 10 was followed by a partially retained intron 10 (the
first 66 nucleotides of this intron were retained) (Figure 5). This resulted from the activation of
a cryptic donor splice site (tg/gt) in intron 10. The sequence of the
881-bp fragment (corresponding to mRNA2 in Table 5) showed that exon 9 joined to exon 11 with the complete
skipping of exon 10 (Figure 5).
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In patient 28 (ga-1 in intron 12) the LDL receptor mRNA was
present only in trace amounts in cultured fibroblasts (Figure 6). RT-PCR of the exon 10 to exon 15
region generated a single fragment of 918 bp in the control and 3
fragments of 899, 759, and 623 bp in patient 28 (Figure 6). The
nucleotide sequence of these fragments (Figure 7) showed that (1) in the 899-bp fragment
(corresponding to mRNA1 in Table 5) exon
12 joined to a partially deleted exon 13 (the first 19
nucleotides of this exon being deleted because of the
activation of a cryptic acceptor splice site, ag/at); (2) in the 759-bp
fragment (corresponding to mRNA2 in Table 5) exon 11 joined to the partially deleted exon 13 (see above);
and (3) in the 623-bp fragment (corresponding to
mRNA3 in Table 5) exon 12 joined to exon
15 with the complete skipping of exons 13 and 14 (Figure 6).
While this study was in progress the same mutation (ga-1 in intron
12) was reported in a Danish FH heterozygote.23
Analysis of the LDL receptor mRNA in the Danish patient
demonstrated the presence of only 2 abnormal mRNAs,24
which were identical to mRNA1 and
mRNA3 (Table 5) found in the present
study.
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We have been unable to investigate the effects of the splicing mutation
found in patient 39, (ga+1 in intron 16) as fibroblasts of this
patient were not available for this type of study. The disruption of
LDL receptor mRNA splicing caused by the mutation found in patient 29
(ga+1 in intron 15) had been previously reported by our
group.11
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Discussion |
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The aim of this study was to characterize the mutations of the LDL receptor gene in a group of patients with the clinical phenotype of homozygous FH; these patients had been identified in Italy during the previous decade and were alive in 1994 or they were born in the period 1994 to 1996. It is likely that our series includes almost all the phenotypically "severe" FH homozygotes who were living in Italy during the time of our study. We are aware, however, of the presence of 3 FH homozygotes living in Sardinia who have not been included in this study, as consent to DNA analysis had not been given. With the exception of patients 2 and 29, reported previously by our group,8 11 and patients 11, 21, and, at least in part, 32, who (after we had performed DNA analysis and LDL receptor assay) were discovered to have been investigated by the Dallas group,1 4 5 the data of the patients included in this study were reported for the first time. Our series included 39 patients, 29 of whom were found to be homozygotes and 10, compound heterozygotes. The mean pretreatment plasma LDL-CH levels were similar in the 2 groups and were of the same order of magnitude as those reported in other large series of FH homozygotes.25 26 27 28 29 In 32 patients we were able to perform LDL receptor assay in cultured fibroblasts and define the residual LDL receptor activity. The finding of an inverse correlation between these 2 parameters is in keeping with a previous study conducted in a group of American FH homozygotes with uncharacterized LDL receptor defects,26 and with recent observations made in French Canadian FH homozygotes with molecularly defined mutations.27 Although the relatively "small" number of patients of our series and their wide age distribution do not allow a statistical analysis, the data about the severity of cardiovascular involvement (as judged by clinical criteria) seem to confirm the idea that the more severe the LDL receptor defect and the higher the plasma LDL-CH levels (as found in patients 19, 20, 21, 28, 30, and 31) (Tables 1
and 3), the more rapid appears to be the rate of progression of
atherosclerosis and the severity of coronary
heart disease.1 30 Although the majority of our patients
were younger than 30 years, we were surprised to find a substantial
number of patients older than 30 years; it is well-known, in fact, that
in the majority of FH homozygotes, if untreated, fatal coronary
heart disease occurs before 30 years of age.1 A similar
observation was reported by Webb et al29 in FH homozygotes
of various ethnic groups. This relatively large proportion of subjects
older than 30 years might reflect a combination of factors such as (1)
a selection bias owing to the sampling, among living FH homozygotes, of
those subjects who had a "relatively mild" LDL receptor defect; (2)
the presence, in some individuals, of other genetic and environmental
factors capable of slowing the rate of progression of
atherosclerosis; (3) the long-term treatment with LDL
apheresis (alone or in combination with hypolipidemic drugs); and (4)
the availability of advanced surgical techniques (such as
coronary artery bypass graft surgery) for the treatment of
coronary heart disease. One important factor in reducing the
rate of progression of coronary atherosclerosis
might have been the use of LDL apheresis, a procedure adopted in
several Italian lipid clinics for the treatment of FH homozygotes.
Several reports indicate that plasma exchange and LDL apheresis improve
the survival of homozygous FH by slowing the progression of
atherosclerosis and promoting the regression of intimal
lesions.31 32 33 34 35 36 LDL apheresis in FH homozygotes would also
reduce the oxidation of LDL, which is thought to play an important role
in the development of intimal lesions.37 At the time of
DNA analysis a dozen of these patients had been being treated
systematically with LDL apheresis for more than 5 years.
Tables 1 and 3 also underscore the difficulty in
predicting the levels of plasma LDL-CH in homozygotes carrying the same
mutation. Although in some patients (19, 20, and 21) plasma LDL-CH
levels were within a fairly narrow range, in others there was a large
interindividual variation. This is in agreement with previous reports
showing a large interindividual variability of plasma LDL-CH levels in
French Canadian FH homozygotes carrying the same
mutation.27 The reasons for this finding still need to be
clarified.
In all homozygotes (Table 2) we were able to identify the
mutations of the LDL receptor gene, by using a combination of Southern
blotting, SSCP analysis, or direct sequencing of the
SSCP-positive region or the complete sequencing of the whole coding
region. In 3 unrelated compound heterozygotes (Table 4) we
failed to identify the mutation in the second allele of the LDL
receptor gene. The failure to identify the second mutant allele in
some FH patients has been reported in other
studies.28 29 38 We cannot exclude the possibility that
mutations located in introns, far from the intron–exon junctions, or
in some regulatory sequences located upstream from the proximal
promoter, have been missed in our analysis of the LDL receptor
gene. One can also argue that some patients, such as patients 35 and 37
(whose residual LDL receptor activity was 20% to 30% of normal
values), were not compound heterozygotes but rather "simple"
heterozygotes with a particularly severe expression of FH. Although
these "relatively high" values of residual LDL receptor activity in
FH homozygotes have been reported by others,29 clinical
and family data make this hypothesis unlikely (see Methods). In the
case of patient 35, we found that all family members of the maternal
line carrying the identified mutant allele (E267X) had lower plasma
LDL-CH levels than the proband and did not manifest the same clinical
phenotype (ie, tendon xanthomas at early age). Because the
proband's father had a moderate elevation of plasma LDL-CH levels, we
favor the idea that patient 35 has inherited a severe mutation
(producing a truncated LDL receptor) from her mother and a relatively
mild mutation from her father. The same considerations can be applied
to patient 37 (20% residual LDL receptor activity), as both parents
had elevated plasma LDL-CH levels. Although only the father carried the
identified mutation found (2-bp deletion in exon 10), the mother, the
putative carrier of the unknown mutant allele, had a brother with
primary hypercholesterolemia (7.02 mmol/L)
and belonged to a family with premature CAD.
Although our data confirm the allelic heterogeneity of FH in Italians as in most European populations, some mutations (D200G, C358R, V502M, G528D, and P664L), found in homozygotes, were found in more than one patient. This observation, as well as the finding that the patients with the same mutation carried the same intragenic haplotype, suggested the presence of a common descent and raised the possibility that these mutations were more frequent in those geographic areas from which these patients came. A systematic screening of 2 mutations (D200G and G528D, found in northern and southern Italy, respectively) among FH heterozygotes living in those geographic areas has led to the identification of 2 large clusters of these mutations in those areas (Bertolini and Calandra, unpublished results).
In this study we report 2 additional examples of FH homozygotes
with splice-site mutations, which allowed us to perform a detailed
analysis of the disruption of LDL receptor mRNA processing
caused by this type of mutation. At present only a small number of
splice-site mutations have been reported in the LDL receptor gene,
mostly in heterozygote subjects.5 11 39 40 41 42 43 44 Here we show 2
splice-site mutations (one in a donor and the other in an acceptor
splice site) that lead to the production of several mRNA
species through the activation of cryptic splice sites in introns
(whereby intron sequences are retained in mature mRNA) as well as in
exons (with partial deletion of the coding sequence) or through the use
of an alternative splicing involving canonical splice sites (exon
skipping). In patient 28 the ga-1 in intron 12 is associated with a
marked reduction of LDL receptor mRNA in fibroblasts and the presence
of 3 abnormal mRNAs. Two of them encode truncated proteins, which are
presumably secreted from the cells, being devoid of the transmembrane
domain, whereas the third abnormal mRNA encodes a receptor with an
in-frame deletion in the epidermal growth factor spacer, a
condition that may reduce the intracellular transport or the recycling
of the receptor.1 The same mutation was recently reported
in some Danish FH heterozygotes.23 24 Nissen et
al24 demonstrated the presence of 2 abnormally spliced
mRNAs (corresponding to mRNA1 and
mRNA3 in Table 5) in
peripheral blood lymphocytes of these patients. The third
abnormal mRNA we found in patient 28 (corresponding to
mRNA2 in Table 5) was not detected in the
Danish patients,24 presumably because this mRNA was
present only in trace amounts in FH heterozygotes. In patient 27
(ga+1 in intron 10) the 2 abnormal mRNAs are predicted to encode 2
receptors: one with an in-frame insertion of 22 amino acids and the
other with an in-frame deletion of 76 amino acids in the epidermal
growth factor precursor homology domain (Table 5). Also in this
case these sequence variations may impair either the intracellular
transport or recycling of the receptor protein. These results, together
with our previous study performed in patient 29,11 confirm
the general idea that mutations involving the invariant
dinucleotides at the 5' or 3' end of introns result almost
invariably in incorrect splicing of the primary transcript, even though
the pattern of aberrant splicing varies in different
genes.45
In conclusion, this study sheds more light on the molecular defects of the vast majority of FH homozygotes in Italy and sets the stage for further studies on the genotype–phenotype relationship and the contribution of other genetic and environmental factors to the progression of premature atherosclerosis in these patients. The data collected in this study have also provided the clue for extending DNA analysis in probands' relatives and for the screening of specific mutations of the LDL receptor gene in defined geographic areas of the country. Finally, the complete clinical and molecular characterization of these patients will provide the basis for a better understanding of the effect of new pharmacological and nonpharmacological treatments that are presently used46 or will be adopted in the future for the therapy of homozygous FH.
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Acknowledgments |
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The authors thank the patients and their families for their collaboration in this study. This study was supported by a grant from the Consiglio Nazionale delle Ricerche (Progetto Finalizzato Invecchiamento) to S.B. and a grant from Istituto Superiore di Sanità to S. Calandra.
The following investigators and institutions also participated in the study: A. Avogaro, Dipartimento di Medicina Clinica e Sperimentale, Università di Padova; B. Capurso, Dipartimento di Geriatria, Università di Bari; C. Martini, Istituto di Medicina Interna, Università di Padova; D. Notarbartolo, Dipartimento di Medicina Interna, Università di Palermo; G.F. Pagano, Dipartimento di Medicina Interna, Università di Torino; A. Postiglione and F. Mancini, Istituto di Medicina Interna, Università di Napoli; C. Sirtori, Istituto di Scienze Farmacologiche, Università di Milano; and C. Stefanutti, Istituto di Terapia Medica, Università di Roma, "La Sapienza."
Received February 17, 1998; accepted August 24, 1998.
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