Pvu II Polymorphism of Low Density Lipoprotein
Receptor Gene and Familial Hypercholesterolemia
Study of Italians
Antonio Daga, Marina Fabbi, Tiziana Mattioni,
Stefano Bertolini, and Giorgio Corte
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Familial hypercholesterolemia is a metabolic disorder inherited as an autosomal
dominant trait characterized by an increased plasma low density lipoprotein (LDL)
level. It has been demonstrated that the disease is caused by several different
mutations in the LDL receptor gene. Although early identification of individuals
carrying the defective gene could be useful in reducing the risk of atherosclerosis
and myocardial infarction, the available techniques for determining the number of the
functional LDL receptor molecules are not sufficiently accurate. The recent isolation
of the LDL receptor gene now makes it possible to use restriction fragment length
polymorphisms to study the inheritance of the defective allele in families with familial
hypercholesterolemia. In the present study, we report the use of a Pvu II restriction
fragment length polymorphism to follow the inheritance of familial hypercholesterolemia in a total of 79 patients from 37 different families. This restriction fragment
length polymorphism allowed unequivocal diagnosis in 32.5% of the cases. Furthermore, in the Italians studied, the absence of a polymorphic Pvu II cutting site
(P1 allele) was found to be strongly associated with familial hypercholesterolemia.
(Arteriosclerosis 8:845-850 November/December 1988)
F
amilial hypercholesterolemia (FH) is a metabolic disorder characterized by a raised plasma low density
lipoprotein (LDL) concentration, xanthomas of skin and
tendons, and premature atherosclerosis. The disease is
inherited as an autosomal dominant trait with a heterozygote frequency of about 1 in 500 in Europeans and
Americans. Heterozygosity is associated with a high risk
of myocardial infarction in the fourth decade of life.
Homozygotes are much more severely affected and often
experience myocardial infarction in the first or second
decade.
Over the past few years, it has been clearly demonstrated that the primary defect in FH is a mutation in the
gene coding for the LDL receptor that prevents a normal
clearance of plasma LDL.1 Several reports have recently
detailed some of these mutations that affect different parts
of the gene. 2 - 5
Early identification of FH heterozygotes, especially in
those cases where the cholesterol level overlaps that of
normal subjects, is highly desirable so that the risk of
future myocardial infarction can be reduced by diet and
life style change. However, the techniques currently available for determining the number of functional LDL recep-
tor molecules on cultured cells are not accurate enough to
give unambiguous results. 678
The recent isolation of a cDNA clone for the human LDL
receptor gene has now made it possible to investigate the
presence of restriction fragment length polymorphisms
(RFLPs), which can be used for early diagnosis of FH. 9
Some RFLPs of the LDL receptor gene that might be used
to follow the inheritance of the affected gene in informative
families have been found. Thus, Humphries et al. 10 - 11 and
Leppert et al. 12 have recently shown that the two alleles
defined by a polymorphic Pvu II site located in the intron
between exons 15 and 16 cosegregate with the disease in
informative families.
In the present study, we report the distribution of the
Pvu II alleles in 112 normolipidemic Italian subjects ages 2 to
99 years old and in 79 FH patients from 37 different families.
Methods
Subjects
The subjects were selected according to the following
criteria: Normolipidemic controls had cholesterol levels
<5.2 mmol/l with LDL cholesterol <3.5 mmol/l (younger
than 30 years old); cholesterol levels s5.7 mmol/l with
LDL cholesterol <4.0 mmol/l (30 years old or older);
triglyceride levels s i . 8 mmol/l; negative family history of
hyperlipidemias, coronary heart disease, or other clinical
signs of atherosclerosis. FH patients had cholesterol
levels s6.7 mmol/l with LDL >4.6 mmol/l (younger than
16 years old); cholesterol levels s7.5 mmol/l with LDL
>5.2 mmol/l (16 years old or older); a total triglyceride
level not exceeding 2 mmol/l; plus two of the following:
tendon xanthomas in the proband or in one or more first
From the Atherosclerosis Prevention Center, the Departments
of Internal Medicine and Biochemistry, University of Genoa, and
the National Institute for Cancer Research, Genoa, Italy.
This work was partially supported by CNR Progetto Finalizzato
Oncologia Grant 85/86.00647.44 and by a Ministero Pubblica
Istruzione grant.
Address for correspondence: Stefano Bertolini, M.D., University of Genoa, Atherosclerosis Prevention Center, Department of
Internal Medicine, Viale Benedetto XV,6, 16132 Genova, Italy.
Received July 14, 1987; revision accepted July 8, 1988.
845
846
ARTERIOSCLEROSIS
V O L 8, No 6, NOVEMBER/DECEMBER 1988
degree relatives, hypercholesterolemic children in the
family, cholesterol levels s7.5 mmol/l in two or more
family members, and family history of coronary heart
disease in one or more first or second degree relatives
younger than 50 years old.
All the subjects gave their informed consent for this
study.
16.5 kb
14
Laboratory Analyses
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DNA was prepared from 10 ml of fresh whole blood by
a sodium dodecyl sulfate (SDS) lysis method.13 DNA was
digested with the Pvu II restriction enzyme, according to
the manufacturer's directions (Bethesda Research Laboratories, Gibco BRL, Scotland). DNA fragments were
separated by size on a 0.8% agarose gel and were
transferred to Gene Screen Plus filters (New England
Nuclear, Du Pont, West Germany) by the Southern blotting technique.14 The LDL receptor probe, pLDLR-2HH1,
kindly provided by David W. Russell, is a 1.9 kb fragment
(bp 1573 to 3486) of the 3' end of the LDL receptor cDNA
clone. The fragment was excised from a 1 % low melting
agarose gel and was labeled by the nick translation
technique with 32p(a)dCTP at 3000 Ci/mmol (Amersham
International, England).13 5 x 106 cpm/ml of the probe
was incubated with filters for 36 hours at 65°C in a 6x
SSC, 1 % SDS, 5 x Denhart, 100 /ug/ml herring DNA
solution. Filters were washed with 0.2 x SSC, 1% SDS at
65°C and exposed to X-ray film (Kodak Xomat AR) for 2 to
4 days at -80°C.
Statistical Analyses
Statistical comparison of allele frequencies was done
by the chi-squared test. The polymorphism information
content was calculated according to the method of
Botstein et al. 15
3.6
Results
As previously described, 1016 digestion with Pvu II generates three different fragment patterns in normal subjects
(Figure 1). One fragment (3.6 kb) is common to all
individuals, while the 16.5 and 14.0 kb derive from two
different alleles (P1 and P2, respectively). The polymorphic Pvu II site has been located in the intron separating
exon 15 and 16 and is due to an adenine-to-guanine
transition in the right arm of the 5' Alu repeat of a cluster
of two tandem Alu sequences.16
The frequencies of the two alleles were determined in
112 unrelated normolipidemic Italian individuals ages 2 to
99 years old (mean age 41.2 years) who had plasma
cholesterol, 4.61 ±0.53 mmol/l; LDL cholesterol, 2.75±0.67
mmol/l; and triglyceride 0.92±0.39 mmol/l. Table 1 reports
the observed frequencies and the distribution of genotypes, which are close to the expected values if the
population is in Hardy-Weinberg equilibrium.
Table 1 also reports a comparison with the frequencies
observed in previous studies in different populations. It is
evident that, in the Italian population, the two alleles are
more evenly represented, since the frequency of allele P2
(0.402) was nearly twice that in the English population
(0.226). Afrikaners17 are, in this respect, more similar to
2.5
Figure 1. Pvu II restriction fragment length polymorphism
detected with the low density lipoprotein receptor probe, LDLR
2HH1. From left to right: P2P2, P1P1, P1P2.
Italians than to English (0.346). Analysis of the allele
frequencies with the chi-squared test showed that the
difference between the Italian and English populations is
statistically significant (#2=11.03, p<0.005).
The Pvu II RFLP was then determined in 79 individuals
with heterozygous FH from 37 different families, ages 3 to
66 years old (mean age 40.9 years) who had plasma
cholesterol, 9.31 ±1.76 mmol/l; LDL cholesterol, 7.38±
1.79 mmol/l; triglyceride, 1.18±0.46 mmol/l. An abnormal
restriction fragment, which was associated with the disease and which could not be assigned to either the P1 or
LDL RECEPTOR GENE POLYMORPHISM
FH2
847
FH3
1
1/1
Daga et al.
1?1
1/1
1
1/2
—
1/1
I
I
1%
I
1/1
FH5
V—-
2/2
1#/1
1/1
171
1/2
B
1/1
172
FH9
FH6
l /
jfc-6
Downloaded from http://atvb.ahajournals.org/ by guest on July 31, 2017
21
FH17
Q - s o
2/2
A6 4
2/2
1%
172
2/2
172
T^
OrBO
1/1 I 172
1/1 I 172
171
172
FH28
•a
1/2
1/2
\
172
FH32
1/1
1?2
1/1
FH 33
jTf-pO
1/2
1/2
^K3
i72
Q - •€>
1/2
I 2/2
1T2
1#/2
2/2
Normal male and female
FH
• O
(heterozygous)
proband
*
^
B ©
Individual deceased
Ml • Myocardial Infarction
0
SD- Sudden Death
Figure 2. Cosegregation of the P1 allele with familial hypercholesterolemia in 17 families.
0
848
ARTERIOSCLEROSIS
VOL 8, No 6, NOVEMBER/DECEMBER 1988
Table 1. Comparison of Genotype Distribution and Allele Frequency of Pvu II Restriction Fragment Length
Polymorphisms in Normoiipidemic Individuals from Different Populations
Number of <illeles (frequency)
Genotype distribution (n)
Italian (n=112)
English (n=62) 1 °
American (n=19) 16
Afrikaner ( n = 6 5 ) 1
P1P1
P1P2
P2P2
40
54
38
20
18
4
29
27
9
P1
134
96
—
85
(0.598)
(0.774)
(0.764)
(0.654)
P2
90 (0.402)
28 (0.226)
— (0.236)
45 (0.346)
Superscripts refer to references.
Table 2. Comparison of Genotype Distribution and Allele Frequency of Pvu II Restriction Fragment Length
Polymorphisms in Normoiipidemic and Familial Hyperchoiesterolemic Individuals
Genotype distribution (n)
Normal controls (n=112)
FH probands (n=35)
Number of alleles (frequency)
P1P1
P1P2
P2P2
P1
P2
40
54
20
15
18
0
134(0.598)
55 (0.786)
90 (0.402)
15(0.214)
FH=familial hyperchoiesterolemic.
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the P2 allele, was observed in two families. The frequencies of the two alleles were estimated in the remaining 35
unrelated patients. As reported in Table 2, FH was strongly
associated with the P1 allele at a frequency of 0.786. The
P1P1 genotype (57.1 % of probands, 58.9% of all patients)
was more frequent than in the normal population (35.7%),
while the P2P2 was not found. Statistical analysis showed
that the frequencies of the alleles in the FH proband group
was significantly different from that of normal controls
(* 2 =8.17, p<0.005).
Only 25 of the 35 probands had a sufficient number of
family members available for study. When the RFLP was
used to follow the inheritance of the LDL receptor gene
in these 25 FH families, the disease was found to
unambiguously cosegregate with the P1 allele in 23
families (Table 3). Figure 2 shows the complete pedigree of seventeen families. Table 4 summarizes the
independent haplotypes found in the 25 families. This
finding had already been reported,10 but was not considered significant because of the low frequency (20%)
of the P2 allele in the English population and the small
number (two) of families examined.
In our study, with the observed frequency of P2 in the
Italian population (0.402, see Table 2), one would expect
that in 40% (10 of 23) of informative families the FH
phenotype would segregate with P2.
Discussion
A number of recent reports indicate that the mutation in
the LDL receptor gene in FH patients is not unique.2"5
Thus, several different mutations, often involving the
multitude of Alu sequences present in the gene, have
been found in different families with FH. However, in most
cases, even several restriction enzymes have failed to
demonstrate any abnormality in the restriction fragments,
either because the deletion involved was too small or
because a point mutation was responsible for the defect.
Thus, in our study, we found an abnormal restriction
fragment inherited with the disease in only 2 of the 37
families examined (data not shown). Both mutations
appeared in a preliminary characterization to be different
from the ones already described. (These will be reported
in detail elsewhere.)
It seems unlikely then, that a restriction pattern characteristic of FH that could be used to unequivocally diagnose
the disease can be found. Nevertheless, once the allele of
the affected gene is identified, the RFLP generated by the
polymorphic Pvu II site between exons 15 and 16 of the
LDL receptor gene can be used to follow the gene's
inheritance. This is especially useful in populations like
the Italian and the Afrikaner, in which the two alleles are
evenly represented. It is, in fact, possible to follow the
inheritance of the defective allele from heterozygous
patients (42.9% in our study) and thus unequivocally
diagnose FH in early infancy in nearly one-third of the
patients (Table 5).
One interesting point is the association between the P1
allele and FH. The single point mutation generating the
extra Pvu II site does not seem, by itself, to adversely
affect the LDL receptor gene function, since the plasma
concentrations of total cholesterol and LDL cholesterol
were not significantly different among the P1P1, P1P2,
and P2P2 groups. Thus, the most likely explanation is that
a mutation causing FH occurred on a chromosome that
lacked the cutting site for Pvu II, and that this mutation
makes a significant contribution to the pool of mutation
causing FH in the Italian population. This would suggest
the existence of a founder effect as is seen in the
Afrikaner population.18
Although Pvu II identifies an individual at risk in only
32.5% of FH cases, it is certainly a step toward an early
and unequivocal diagnosis of this disease. If different
RFLPs of the LDL receptor that can be used to split the
Pvu II alleles are identified, it will be possible to more
accurately follow the inheritance of the disease and to
eventually offer an early diagnosis in most, if not all,
families with FH. Some promising RFLPs have, indeed,
been recently reported with Ava II, Apa LI, and
NCO
LDL RECEPTOR GENE POLYMORPHISM
Table 3.
849
Daga et al.
Cosegregation of Familial Hyperchoiesterolemia with P1 Allele in 25 Families
Offspring
Parent
Family
Unaffected
Affected
FH2
P1P1 (sister)
FH3
FH5
P1P1 (sister)
P1P1 (brother)
P1P1 (sister)
P1P2
P1P1
P1P2
P2P2
FH7
FH9
FH11f
FH13
FH14
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FH15
FH17
FH20
FH21
FH22
FH24
FH25
FH27
FH28
FH29
FH31
FH32
FH33
FH34
FH35
P1P1(1)
P1P1(1)*
P1P1(1)
P1P1(1),P1P2(1)
P1P1(1)*
P1P1(1)*
P1P1(2)
P1P1
P1P2
P1P1
deceased
P1P2
deceased
deceased
P1P1
deceased
P1P1
P1P2
P1P2 (sister)
P1P1 (sister
P1P1
P1P1
P1P1
P1P2
P1P1
P1P1
P1P1 (sister)
FH10t
P1P1(1), P1P2(1)
P1P2(1)
P1P1
P1P1
P1P1
P1P1
P1P2
P1P2
P1P1 (cousin)
P1P2 (cousin)
P1P2
P1P1
FH6
Unaffected
Affected
P2P2(1)
P1P2(1)
P1P2(2)
P1P1(2)
deceased
P2P2
deceased
P2P2(3)
P2P2(2)*
P1P2(3)*
P1P2(1)
P1P1(1)*
P1P1
P1P2(2)
P2P2
P1P2
P1P2(1)
P1P1(1)
P1P1(1)
P1P1
P1P2
P1P1
P1P1(1)
P1P2(1)*
P1P1(1)
P1P1
P1P2
P1P2
P1P1(1)
P1P2(1)
P1P2(1)
P1P1(1)
P1P2(2)*
P1P1(1)
P1P2
P1P1 (sister)
deceased
(brother)
P1P1 (sister)
P1P1
P1P1
P1P2
P1P1
P1P2
P1P2
P2P2
P1P2
P1P1(1), P1P2(1)
P1P1
deceased
P1P1(1)*
P1P1(1)
P1P2
P1P2
P1P1(1), P1P2(1)
P1P1(1), P1P2(1)
P2P2(1)*
P1P2(1)*
P1P2(1)
The number in parenthesis is the number of subjects.
'Cases in which Pvu II RFLP allows unequivocal diagnosis of health or disease without knowledge of plasma cholesterol level.
tAmbiguous families.
Acknowledgments
We thank David W. Russell, Michael Brown, and Joseph
Goldstein for supplying the LDL receptor probe.
in 25
Table 4.
Families
Independent Haplotypes Observed
D-P1
D-P2
D-?
d-P1
d-P2
d-?
0
2
58
39
26
2
23
D=the disease allele; d=the normal allele.
Table 5.
Probability that Offspring Are Informative for Familial Hyperchoiesterolemia in Italian Population
Genotype frequency
of affected parent
P1P1
P1P2
P1P2
P1P2
Total
(0.571)
(0.428)
(0.428)
(0.428)
Informative offspring
Genotype frequency
of unaffected mate
Frequency
of mating
All genotypes (1)
P1P1 (0.357)
P1P2 (0.482)
P2P2 (0.161)
0.5711
0.1528
0
100
0.2063
0.0689
50
Polymorphism information content value=0.3248.
Frequency
100
0
0.1528
0.1031
0.0689
0.3248
850
ARTERIOSCLEROSIS
VOL 8, No 6, NOVEMBER/DECEMBER 1988
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Pvu II polymorphism of low density lipoprotein receptor gene and familial
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A Daga, M Fabbi, T Mattioni, S Bertolini and G Corte
Arterioscler Thromb Vasc Biol. 1988;8:845-850
doi: 10.1161/01.ATV.8.6.845
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