© 1997 American Heart Association, Inc.
Articles |
Familial Moderate Hypercholesterolemia Caused by Asp235
Glu Mutation of the LDL Receptor Gene and Co-occurrence of a De Novo Deletion of the LDL Receptor Gene in the Same Family
From the Departments of Medicine (U.-M.K., H.G., T.A.M., K.K.) and of Pharmacology and Toxicology (U.-M.K.), University of Oulu, and Department of Medicine (K.K.), University of Helsinki (Finland).
Correspondence to Kimmo Kontula, MD, Associate Professor of Medicine, Department of Medicine, University of Helsinki, FIN 00290, Helsinki, Finland.
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Abstract |
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Abstract We identified a large family in which a hitherto unreported point mutation of the LDL receptor gene (Asp235
Glu) cosegregated with moderately elevated serum LDL
cholesterol concentration. Within one generation, the mean
serum total and LDL cholesterol levels in four heterozygous
carriers of this mutation (7.76±1.46 and 5.89±1.56 mmol/L,
respectively) were significantly (P<.05) higher than the
corresponding concentrations in their five nonaffected siblings
(5.81±0.57 and 3.77±0.54 mmol/L, respectively). Lipid levels in
carriers of the Asp235Glu mutation were, however, markedly lower
than the corresponding total and LDL cholesterol levels
(about 12 and 10 mmol/L, respectively) in heterozygous patients
with the two common LDL receptor mutations (FH-Helsinki and FH-North
Karelia). None of the four siblings in the age range of 54 to 69 years
had experienced a myocardial infarction, although symptoms suggestive
of coronary artery disease were present in two and tendon
xanthomas were found in one. Expression of the mutant receptor in COS
cells indicated an approximately 50% to 70% reduction of LDL-binding
activity compared with the normal receptor. One patient (female, aged
39 years) had severe hypercholesterolemia in
the range of 13 to 20 mmol/L when untreated, extensive
coronary artery disease as demonstrated by angiography, and
extensor tendon xanthomatosis. In addition to the Asp235Glu
mutation, she was found to have a de novo deletion of exons 14 and 15
in her other LDL receptor allele. In this subject, the total LDL
receptor activity of mitogen-stimulated blood lymphocytes was very low.
In conclusion, along with another LDL receptor gene mutation (FH-Espoo
or deletion of exon 15) described by us previously, the Asp235Glu
mutation (designated as FH-Keuruu) indicates that moderate varieties of
inherited hypercholesterolemia may result from
LDL receptor gene mutations of mild expression.
Key Words: LDL receptor gene mutation • familial hypercholesterolemia
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Introduction |
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Familial hypercholesterolemia, or FH, an autosomal codominant disorder of lipoprotein disorder with a prevalence of about 1 in 500, results from mutations of the LDL receptor gene.1 At the DNA level, FH is extremely heterogenous, and by 1992 more than 150 different LDL receptor mutations had been described.2 When present in a heterozygous form, most of the mutations identified thus far have been reported to cause a typical clinical picture of FH, with grossly elevated serum LDL cholesterol levels, tendon xanthomas, and premature coronary heart disease.
Patients with homozygous FH have been shown to have considerable interindividual variation in serum cholesterol levels as well as in the expression of coronary heart disease,3 which in one study was directly attributed to the nature of the causative mutation.4 In heterozygous FH, common genetic variation of gene loci other than the LDL receptor gene, such as those encoding apolipoprotein B,5 6 apolipoprotein E,7 8 9 10 11 12 13 and lipoprotein lipase,14 has been suggested to contribute to the clinical expression of the disease. Common variation of the intact LDL receptor allele may also exert subtle influence on serum cholesterol levels in patients with heterozygous FH.15 In addition, relatively mild clinical features of patients with Chinese heterozygous FH with receptor-negative mutations were postulated to be explained by dietary and other environmental factors prevailing in that particular population.16
There is, however, evidence that the particular type of the underlying LDL receptor gene mutation may affect serum lipid levels17 18 and their response to treatment with HMG-CoA inhibitors,19 20 even in patients with the heterozygous form of FH. Two recent studies21 22 directly demonstrated the occurrence of mutations of the LDL receptor gene that cause a "mild" clinical phenotype of heterozygous FH. The Finnish FH-Espoo mutation selectively deletes exon 15 of the LDL receptor gene, encoding the carbohydrate-containing extracellular domain of the receptor protein, and results in a moderate variety of inherited hypercholesterolemia,21 and the FH Afrikaner-3 mutation, substituting Asn for Asp at amino acid 154 in the ligand-binding domain of the LDL receptor protein,22 was shown to be associated with lower serum cholesterol levels and less clinical severity than another founder mutation (FH Afrikaner-2) prevalent in the same population studied. The present work adds an important adjunct to these previous findings to raise the idea that "mild" mutations of the LDL receptor gene occur in the population and also describes a rarely occurring de novo mutation of the LDL receptor gene in the same family.
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Methods |
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The Proband and Her Family
The proband, a woman born in 1956, was examined because of extensor tendon xanthomatosis at the Lipid Outpatient Clinic of the University Hospital of Helsinki in 1976. Her serum total cholesterol level from 1976 to 1982 varied between 13 and 20 mmol/L with a mean value of 17.5 mmol/L. Her serum HDL cholesterol level was 0.7 to 1.3 mmol/L, and her serum triglyceride level about 1 mmol/L. Cholestyramine, nicotinic acid, or clofibrate, alone or in combination, resulted in unsatisfactory hypolipidemic responses. Symptoms suggesting angina pectoris developed, and coronary angiography carried out in 1985 disclosed three-vessel coronary artery disease. Treatment with HMG-CoA reductase inhibitor and resin was instituted, and in 1991, while on lovastatin 80 mg/d and colestipol 10 g/d, her serum cholesterol level was 5.6 mmol/L, her HDL cholesterol level was 0.72 mmol/L, and her triglyceride level was 0.65 mmol/L. Since then her condition has been relatively good, with occasional exertion angina still present. Late in 1995 she became pregnant, and she delivered a healthy boy in July 1996. She gave her informed consent to carry out DNA analysis of the cord blood.
The proband has four sisters and one brother, all in good health (Fig 1
). The father of the proband, born in
1924, is healthy, and his serum cholesterol level is normal
(5.6 mmol/L). The mother of the proband (Fig 1), born in
1926, is in relatively good health. She has no angina or claudication,
but a submaximal ergometer test performed in 1987 showed a 2-mm
depression of the ST segments in the anterior
electrocardiogram leads; she is currently on
hypolipidemic drugs and being followed up by a general
practitioner. The oldest sister of the proband's mother
died of gastric cancer, but all the remaining siblings (four sisters,
four brothers) were available for DNA studies and analysis of
serum lipid levels (Fig 1).
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DNA Analysis
Details on the screening for gross rearrangements in the LDL
receptor gene by Southern blot analysis are given in another
report.23 For screening of minor gene rearrangements, all
18 exons of the LDL receptor gene were individually amplified from
genomic DNA by PCR, and SSCP analysis was performed essentially
as described by Orita et al.24 PCR primers contained the
same intron sequences of the LDL receptor gene as those published by
Leitersdorf et al.25
The exon 5 variant was further characterized by direct sequencing of the double-stranded PCR product according to a modification of the dideoxy chain termination method.26 Screening for the mutation in other family members and unrelated hyperlipidemic individuals was carried out by NciI restriction enzyme digestion of amplified exon 5, followed by analysis on 2% agarose gels.
A search for the presence of the deletion allele (deletion of exons 14 and 15) in the child of the proband was carried out by a PCR technique. DNA was amplified using the primers25 SP78 (5'-GTCATCTTCCTTGCTGCCTGTTTAG-3') and SP85 (5'-CGCTGGGGGACCGGCCCGCGCTTAC-3'). Under these conditions, the normal allele does not result in visible amplification products, and the deleted allele generates a fragment of approximately 1800 bp, permitting its identification upon electrophoresis on 1% agarose.
RNA Amplification and Sequence Analysis
Total RNA was isolated27 from fibroblasts obtained
from a skin biopsy specimen of the patient with the deletion.
First-strand cDNA synthesis was accomplished by extension with primer
3588 (5'-ATCCCAACACACACGACAGA-3'), complementary to normal exon 18
sequence. One-half of the reverse-transcription mixture was then
included into a 50-µL PCR with 50 ng each of primers 91483
(5'-GTTCTTAAGCCGCCAGTTCT-3') and 91363
(5'-AGTGCCAACCGCCTCACAGG-3'), complementary to the normal exon 17
and exon 13 LDL receptor sequences, respectively. PCR was carried out
for 40 cycles with a temperature profile of 95°C, 55°C, and 72°C
for 1 minute each. The amplification product corresponding to the
deleted mRNA was isolated from a 2% agarose gel and purified by Qiaex
gel extraction according to the supplier's protocol. Sequence
determination was carried out by direct sequencing of the PCR
product as described above for genomic DNA.
Plasmid Construction and Expression of the Mutant LDL Receptor
cDNA
The cDNA encoding the LDL receptor was subcloned into the
phagemid pALTER-1. A point mutation of GAC (Asp 235) to
GAA (Glu) was introduced into single-stranded phagemid DNA
using the mutagenic oligonucleotide 3874
(5'-ATATTCCCGTTCACACTGCC-3') according to the supplier's protocol
(Altered Sites In Vitro Mutagenesis Kit).
Oligonucleotide primers 5885 (5'-TCAGACGAGGCCTCC3') and
91167 (5'-TCTTAAGGTCATTGCAGACG-3'), complementary to exon 4 and exon 7
sequences, respectively, were used to generate the PCR product for
restriction enzyme analysis. The mutated LDL receptor cDNA was
then ligated into the Xba I and Sac I sites of
the SV40 promoter-based expression vector pSVL for use in the transient
transfection experiments.
COS cells were transfected with the normal and mutant LDL receptor cDNAs using the liposome method and Lipofectamine reagent. Cells were assayed for the binding of increasing concentrations of 125I-labeled LDL 48 to 72 hours later at 4°C. The amount (ng/dish) of 125I-LDL bound at each ligand concentration by cells mock-transfected with the expression vector pSVL was subtracted from the value obtained with a given plasmid. This value was divided by the amount of LDL receptor DNA per dish, as assayed by slot-blot hybridization, to normalize for transfection efficiency.
Determination of the Activity of LDL Receptors on
Lymphocytes
The functional LDL receptor assay was carried out using the
technique of Cuthbert et al28 as described
previously.21 In this assay, the activity of the LDL
receptor on mitogen-stimulated lymphocytes is evaluated in vitro under
conditions in which endogenous cholesterol
biosynthesis is blocked by lovastatin to render cellular
proliferation dependent on exogenous LDL. Curves indicating cell
proliferation rate are plotted against increasing concentrations of LDL
and used to compare individual samples. A blood sample from the proband
was obtained while she was pregnant and on colestipol only.
Assays for Serum Lipid Levels and Lipoprotein Kinetics
Serum lipoproteins were separated by
ultracentrifugation into density classes, as described
in the Manual of Laboratory Operations of the Lipid Research
Clinics Program,29 to VLDL, IDL, LDL, and HDL. Serum and
lipoprotein cholesterol and triglyceride and
apoprotein B were measured automatically using commercial kits.
For the kinetic studies, 50 mL of fasted EDTA plasma was drawn, from which autologous LDL was separated by serial preparative ultracentrifugations and iodinated with 125I by a modified iodine–monochloride method.30 31 Three days before the injection, subjects started to take peroral potassium iodide. Approximately 1 mg of the labeled LDL was mixed with 5% human serum albumin, filtered, and injected intravenously. The total amount of radioactivity did not exceed 20 µCi. After the injection, blood samples of 10 mL were collected and counted for 14 days. The die-away curves were constructed for 125I–LDL. FCR for LDL was determined using a two-pool model.32 TR was determined by multiplying FCR by the pool size, which was calculated by multiplying the apolipoprotein B plasma concentration by plasma volume, which in turn was estimated to be 4.5% of body weight.
Statistical Analyses
Differences in the mean serum lipid levels in mutation carriers
and noncarriers were analyzed using the Student's
t test.
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Results |
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Serum Lipid Levels in Family Members
The pedigree of the family is illustrated in Fig 1
. Moderate
hypercholesterolemia (>6 mmol/L)
was present in several members of the family, and the proband had
severe hypercholesterolemia. Achilles tendon
xanthomatosis was present in the proband and subject I-6 (Fig 1)
but in no other family members. The occurrence of coronary
heart disease was suspected in subjects I-3 and I-4 and documented in
the proband (II-5). Serum HDL cholesterol and
triglyceride levels were essentially normal in all the
family members (Table 1).
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Identification of Two LDL Receptor Gene Mutations in the
Family
Southern blot analysis of the proband's DNA sample, using
a cDNA probe encompassing exons 11 to 17 of the LDL receptor gene and
restriction enzyme BamHI, Xba I, or
Pvu II, revealed aberrant hybridization signals in each case
(data not shown), suggesting the presence of a major rearrangement in
the 3' portion of one of her LDL receptor alleles. The
hybridization pattern displayed by the proband's DNA was clearly
different from that produced by a DNA sample from a heterozygous
carrier of the Finnish founder gene FH-Helsinki, which deletes exons 16
and 17 and part of exon 1833 ; overall, Southern blotting
data suggested deletion of exons 14 and 15. To demonstrate the exact
nature of this mutation, a partial cDNA molecule corresponding to the
mutant allele was produced by reverse transcription and PCR
amplification of fibroblast RNA from the proband. DNA sequencing showed
that exons 14 and 15 were indeed deleted, and there was a direct
continuum of the DNA sequence from exon 13 to exon 16, with an apparent
maintenance of the reading frame in exons located 3' of the
deletion point (Fig 2).
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The deletion eliminating exons 14 and 15 was not found in the DNA
samples from the mother or father of the proband (Fig 1). Paternity
tests using five different polymorphic DNA markers (MCT118, YNZ22,
HUMTH, D3S, vWA, and FES) disclosed a paternity index of 3946
corresponding to a 99.97% likelihood of paternity (data not shown).
These data demonstrate that this mutation (deletion of exons 14 and 15)
arose on a de novo basis.
Because of the severe
hypercholesterolemia present in the proband
(Fig 1), homozygosity for a defect in the LDL receptor gene was
initially suspected in her case. Therefore, all 18 exons of the LDL
receptor gene were screened using an SSCP technique. An aberrant band
shift was disclosed when exon 5 was analyzed using this
technique (Fig 3A). Direct sequencing of
the amplified exon 5 revealed a C to A substitution at
nucleotide 768, consistent with a change of the
codon 235 from GAC to GAA (Fig 3B). This substitution is predicted to
substitute glutaminic acid for aspartic acid at position 235, which is
located in the sixth cysteine-rich repeat of the ligand-binding domain
of the LDL receptor protein.34 The proband was thus
considered to most likely represent a compound heterozygote,
bearing an Asp235Glu mutation in one and a deletion of exons 14 and
15 in the other LDL receptor allele (Fig 1). Results from the
functional LDL receptor studies were consistent with this
assumption. Thus, while cells from healthy control subjects
proliferated efficiently when the LDL concentration was increased, to
overcome the block in cholesterol biosynthesis, close to 1
µg/mL, cells from the proband grew only poorly under the same
conditions (Fig 4). Our previous studies
indicated that the extent of inhibition of cell proliferation by
lovastatin was approximately 50% when lymphocytes from
heterozygous patients with the FH-Helsinki mutation were examined under
similar conditions.21
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Mutation Analysis of the Family Members
All family members of the proband were subsequently screened for
the presence of the Asp235Glu mutation using an assay combining
amplification of exon 5 by PCR, followed by digestion of the
amplification products with the restriction enzyme NciI.
Amplification of the normal exon 5 under the conditions chosen
generates a 173-bp DNA fragment that is cleaved into 100- and 73-bp
fragments by NciI (Fig 5A).
The C768A mutation eliminates an NciI restriction site
normally present in exon 5, thus permitting an easy means of
mutation detection. The Asp235Glu mutation was identified in not
only the proband but also her mother and four other family members (Fig 1). In addition, analysis of an umbilical cord blood sample
obtained after delivery of the proband's child revealed that the
Asp235Glu mutation (Fig 5A), but not the deletion of exons 14 and 15
(Fig 5B), was present in the child, thus unambiguously
demonstrating that these two mutations were present in different
LDL receptor alleles of the proband.
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phage DNA cut with
HindIII); lane 2, mother of the proband; lane 3, proband;
lane 4, father of the proband; lane 5, child of the proband; and lane
6, PCR control (buffer). The arrow (with an asterisk) shows the
position of the 1.8-kb fragment amplified in the sample of the proband
(lane 3).
Pedigree analysis demonstrated a suggestive, although not
absolute, cosegregation of the Asp235Glu mutation with moderate
hypercholesterolemia (Fig 1). Within the older
generation examined, the mean serum total and LDL
cholesterol levels in the four heterozygous carriers of the
Asp235Glu mutation (7.76±1.46 and 5.89±1.56 mmol/L,
respectively) were significantly (P<.05) higher than the
corresponding concentrations in their five nonaffected siblings
(5.81±0.57 and 3.77±0.54 mmol/L, respectively). Serum
lipid levels in the proband were highly variable but were, when
measured under drug-free conditions, mostly markedly higher than in a
typical heterozygous FH patient (see "Methods").
Thirty unrelated subjects with moderate to severe
hypercholesterolemia and 46 unrelated subjects
with a clinical diagnosis of FH but without either of the two Finnish
founder mutations (FH-Helsinki or FH-North Karelia)35 were
screened for the presence of the Asp235Glu mutation. No additional
carrier of this gene was found. The mutation was subsequently
designated as FH-Keuruu for the birthplace of the proband's
mother.
Expression of the Asp235Glu Mutant Allele In Vitro
COS cells were transfected with either the wild-type or the
C768A mutated LDL receptor cDNA and subsequently analyzed
for binding of 125I-labeled LDL. In each case, increasing
concentrations of 125I-labeled LDL demonstrated saturable
and specific binding sites for LDL, but the maximal binding capacity in
the cells transfected with the mutant cDNA, when normalized to the
transfection efficiency, was only approximately 30% to 50% of that of
the cells transfected with the wild-type cDNA (Fig 6).
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Studies of LDL Kinetics In Vivo in the Family
LDL apoprotein B kinetics was studied in the proband (II-5), her
mother (I-3), father, and brother (II-2), as well as in two affected
(I-4 and I-6) and one unaffected (I-5) uncles (Fig 1, Table 236 ). In the proband, the
concentration of LDL apoprotein B was very high, and the LDL
cholesterol/LDL apoprotein B ratio was much lower
than the corresponding ratio in the other family members and control
subjects (Table 2). In addition, the proband was characterized by a
very low FCR and a high TR of LDL (Table 2). There was a trend toward
lower FCR and higher TR in the Asp235Glu mutation carriers than in
the noncarriers, but subject I-5, a noncarrier, was an exception to
this rule (Table 2).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This report describes two hitherto unreported mutations of the LDL receptor gene that both show special features: one (deletion of exons 14 and 15) appears to have arisen on a de novo basis, and the other (FH-Keuruu or Asp235
Glu) is associated with only moderate elevation
of serum LDL cholesterol level. When present
simultaneously in a compound heterozygous person, these
mutations were associated with a clinical phenotype
intermediate between a homozygous and a heterozygous FH subject. There is only one previous report of de novo mutations of the LDL receptor gene. Kotze et al37 described a newly arisen mutant LDL receptor gene with a duplication of 18 bp in exon 4. Although a number of large deletions eliminating exons 14 and/or 15 have been reported in earlier studies,2 the particular type of deletion characterized in this article has not been described previously, even if its exact mechanism cannot be determined. A transcript molecule encoded by the mutant allele was present in fibroblasts of the proband, but there is no proof that a corresponding protein product, lacking the 108 amino acids encoded by exons 14 and 15, is present on the cell membrane. In addition, we cannot exclude the possibility that the deleted gene could code for additional receptor forms derived by alternative splicing of the primary transcript.
During the past few years we have identified 17 different mutations of
the LDL receptor gene in Finland.21 23 33 35 38 39 40 Except
for the four common mutations (FH-Helsinki, FH-North Karelia, FH-Turku,
and FH-Pori), all seem to be present in single families only, and
all except one rare mutation (Gly457Arg or FH New York-2) are unique
to the Finnish population.39 The FH-Keuruu (Asp235Glu)
point mutation described here has not been reported previously. The
results of our initial screening studies suggest that it is not a
common founder gene causing elevated serum cholesterol
levels among the Finns. Attempts to search for its presence among
moderately hypercholesterolemic subjects of other
populations are facilitated by the convenient PCR assay described in
this report. We have previously shown that deletion of exon 15 alone
(FH-Espoo) is associated with partial deterioration of LDL receptor
functioning and a mild form of FH.21
The Asp235Glu mutation occurs in the sixth cysteine-rich repeat in
the ligand-binding region of the LDL receptor protein.34
This mutation is conservative in nature, with substitution of a
negatively charged amino acid for another with similar physicochemical
characteristics, which may explain the relatively mild phenotypic
consequences of this DNA alteration. Expression of an LDL receptor cDNA
corresponding to the FH-Keuruu gene in COS cells suggested a loss of
approximately 50% to 70% of receptor activity. A mutation chemically
identical to the FH-Keuruu mutation but present in a nearby codon
(Asp245Glu or FH Cincinnati-1) is likewise associated with some
residual (15% to 30%) receptor activity.2 Interestingly,
another point mutation in the same codon as FH-Keuruu, changing
aspartic acid to the neutral amino acid glycine (Asp235Gly or FH
Nevers), was found to result in an LDL receptor with only 5% to 15%
of activity compared to the normal one.2 Our data do not
allow us to attribute deterioration of the FH-Keuruu receptor activity
to a binding-defective allele (functional class 3) or
transport-defective allele (functional class 2). In fact, most of
the point mutations in exons 4 to 6 of the LDL receptor gene were shown
to represent class 2B (partial defect in transport between the
endoplastic reticulum and Golgi apparatus)
mutations.2
Serum total and LDL cholesterol levels in the carriers of
the FH-Keuruu mutation were approximately 30% and 50% higher,
respectively, than the corresponding levels in noncarriers of the same
family. These concentrations in the FH-Keuruu carriers were, however,
markedly lower than the corresponding total and LDL
cholesterol levels (about 12 and 10 mmol/L,
respectively) in heterozygous patients with the two common LDL receptor
mutations (FH-Helsinki and FH-North Karelia) studied previously by
us.35 In our proband, with two different mutations of the
LDL receptor gene, slow catabolic rate of LDL and efficient synthesis
of apolipoprotein B seem to explain the very high levels of LDL
cholesterol and apoprotein B in serum (Table 2). In this
subject the ratio of cholesterol to apoprotein B in LDL
particles was low (Table 2), suggesting that LDL particles were small
and dense, which may explain the aggressive behavior of her heart
disease. Although the FCRs of the LDL particles of the remaining
heterozygous carriers of the FH-Keuruu gene tended to be lower than in
noncarriers, these changes were not consistent (Table 2).
Until now, analyses of naturally occurring mutations of the
human LDL receptor gene have concentrated on DNA alterations resulting
in typical severe forms, whether homozygous or heterozygous, of FH.
Only a few studies have systematically addressed the possibility of
phenotypic variation according to the mutation type of the LDL receptor
gene. Studies among young French Canadian patients with homozygous FH
indicated that the average plasma cholesterol level was
markedly higher and that coronary death was more frequent and
occurred at an earlier age in patients with the 10-kb promoter area
deletion than in those with the Trp66Gly point
mutation.4 Subtle differences in serum lipid levels and
their responses to statin treatment were observed, first, between
heterozygous FH patients with different classes of founder mutations
(Lebanese, Sephardic, and Lithuanian) in Israel20 and
second, between heterozygous FH patients with any of the three founder
genes (FH-Afrikaner-1, -2, and -3) in South
Africa17 19 22 ; however, within each mutation class cited,
serum LDL cholesterol levels were relatively high and in
the range of 7.5 to 9 mmol/L. Recently, a single-base
substitution at the proximal Sp1 binding site of the LDL receptor gene
promoter was reported to be associated with only moderately elevated
serum cholesterol levels, but the patient still had two
myocardial infarctions at a young age.41 The Finnish
"mild" LDL receptor mutations, the FH-Espoo characterized
previously by us21 and the FH-Keuruu described here,
appear to show an even lower degree of phenotypic expression, with the
mean serum LDL cholesterol level in heterozygotes in the
range of 6 to 7 mmol/L. At the full extreme of the end
toward mild mutations of the LDL receptor gene, Gudnason et
al42 showed that the relatively common Ala370Thr
polymorphism of the LDL receptor does not result in measurable
differences in the LDL receptor function in vitro and is associated
with only marginal variation of serum LDL cholesterol
levels in vivo.
In conclusion, the present work, along with previous studies addressing genotype-to-phenotype relationships in FH, shows that the mutation type of the LDL receptor gene may have profound influences on in vivo LDL receptor functioning. The significance of subtle LDL receptor mutations as determinants of serum LDL cholesterol variation at the population level still remains an issue requiring further research.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by research grants from the Medical Council of the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cultural Foundation, the University of Helsinki, the Paulo Foundation, the Paavo Nurmi Foundation, and the Finnish Heart Association. We thank Kaija Kettunen for expert technical help.
Received March 18, 1996; accepted October 9, 1996.
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