Download Characterization of Deletions in the LDL Receptor Gene in Patients

762
Characterization of Deletions in the LDL
Receptor Gene in Patients With Familial
Hypercholesterolemia in the United Kingdom
Xi-Ming Sun, Julie C. Webb, Vilmundur Gudnason, Steven Humphries, Mary Seed,
Gilbert R. Thompson, Brian L. Knight, and Anne K. Soutar
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A sample of 200 patients with a clinical diagnosis of heterozygous (189) or homozygous (11) familial
hypercholesterolemia (FH) attending lipid clinics in the London area have been screened for the presence
of major gene defects in the low density lipoprotein (LDL) receptor gene by Southern blotting of genomic
DNA with specific probes. This study Is part of a project to determine the frequency of known mutations
in the LDL receptor gene in this population. A new polymorphism for the enzyme Bgt II was identified by
hybridization with a probe specific for the promoter plus exon 1 of the LDL receptor gene. The observed
frequency of the rare allele, characterized by a Bgi II fragment of 13 kb compared with 10 kb for the
common allele, was 0.08 in this group of FH patients. Several individuals who were heterozygous for the
rare allele were also heterozygous for a mutation elsewhere in the LDL receptor gene that is known to
cause FH. Eight different mutations, seven deletions and one duplication, were detected in a total of nine
patients, accounting for 4.5% of the mutant alleles in this group. Three of the mutations are apparently
identical to deletions that have been described previously in FH patients of British or European origin,
while the remaining five have not been described. Two of these were in patients of Polish and Asian Indian
origin, while the other three were in patients of apparently British ancestry. (Arteriosclerosis and
Thrombosis 1992;12:762-770)
KEY WORDS • atherosclerosis • polymorphisms • mutations • genetic screening • hyperlipidemia
F
amilial hypercholesterolemia (FH) is an inherited disease caused by mutations in the gene for
the low density lipoprotein (LDL) receptor that
affects approximately one person in every 500 in most
populations, making it one of the more common inherited diseases of metabolism.1 Individuals in whom one
LDL receptor gene is defective have a plasma cholesterol concentration approximately twice that of the
mean of the whole population, and as a result, excessive
amounts of cholesterol are deposited in extrahepatic
tissues. This leads to the formation of xanthomas,
characteristically in tendons, and to accelerated atherosclerosis, with the result that heterozygous FH patients
frequently suffer from premature coronary heart disease. Homozygous individuals, with two defective
genes, are more severely affected and rarely reach the
age of 30 unless stringent methods to reduce plasma
cholesterol are applied.2 Homozygous FH is rare and
clinically unmistakable, but it is often not possible to
make an accurate diagnosis of heterozygous FH on the
From the MRC Lipoprotein Team (X.-M.S., J.C.W., G.R.T.,
B.L.K., A.K.S.), Hammersmith Hospital; the Department of Medicine (V.G., S.H.), University College and Middlesex School of
Medicine; and the Department of Medicine (M.S.), Charing Cross
and Westminster Medical School, London, UK.
Supported in part by grants from the British Heart Foundation
(grants No. 89/14 and RG5). X.-M.S. is a visiting fellow sponsored
by the British Council.
Address for reprints: Dr. A.K. Soutar, MRC Lipoprotein Team,
Hammersmith Hospital, Ducane Road, London W12 0HS, UK
Received February 25, 1992; accepted March 24, 1992.
basis of clinical criteria alone; many hypercholesterolemic patients can only be described as "probable FH"
or even "possible FH" unless they have tendon xanthomas or a well-defined family history of hypercholesterolemia and premature coronary heart disease.
For this reason a diagnostic test based on the identification of the specific mutation in the LDL receptor
gene would prove invaluable, permitting an accurate
diagnosis on which treatment and counseling could be
based. However, the marked heterogeneity of FH at the
level of the gene precludes the development of any
simple screening test at present, and the mutation in
each patient has to be determined de novo. It has
recently been estimated that there are at least 180
different defective alleles of the LDL receptor gene in
known homozygous FH patients from many different
parts of the world, only a few of which have been
characterized in terms of the underlying mutation.3 On
the other hand, in some isolated populations a founder
effect has resulted in a higher-than-normal frequency of
FH patients in whom the majority have the same or one
of a small number of mutant alleles. Examples of these
include the Lebanese,4 French Canadians,5 Afrikaners
in South Africa,6 and Jewish individuals of Lithuanian
origin.7 Although it is unlikely that a predominant
founder gene effect will be observed in more heterogeneous populations of FH patients, such as that in the
United Kingdom, it is possible that a small number of
mutations may be relatively common, particularly in
groups of similar cultural or ethnic background. How-
Sun et al
4 3 6
78 910
1112
1314
LDL Receptor Gene Deletions in UK
5Kb
16 17
15
763
It
(a)
exooil2-15 probe («mpllflml 400bp cDNA fragmenl)
= ^
T
ft»mHI funfiiiwit f
1913 bp probe
(b)
1700bp probe
(excm 1 - 11)
(excm 11-18
+14bpofexon 10)
14.0
(c)
I
I U 10/13
I 2.5 |[
13
9.5
3.5 J
21
EcoRV/Bgffl
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1
15
8.5
9
19
15
X
9
a.7i
10
promoter probe
X
ffl*
6
1
1
II 1
6
11
1
XXX
X
EcoRV/Xbal
EcoRV/SuI
J
Kpnl
Kpol/Xbal
EcoRI
eiont 12-15 probe
1700 probe
1913 probe
InSvidnal erocupedfic proba (mpUfled gene fragmeoii) not shown
FIGURE 1. Diagram of the structure of the low density lipoprotein receptor gene14 (panel a) and its cDNA (panel b), showing
positions of restriction enzyme sites employed for the production of cDNA probes, the 5' region of the gene amplified to provide a
"promoter" probe, and restriction enzyme maps of the gene indicating the size (kb) of the expected fragments with each probe
(panel c). Polymorphic sites for Pvu II'5 and Bgl / / (this article) are marked by an asterisk. E, Eco RV; B, Bgl //; X, Xba /; 5,
Sac /; K, Kpn /.
ever, no systematic survey of the frequency of different
mutations in the LDL receptor gene in the FH population in the United Kingdom has been undertaken.
Therefore, we have selected a group of 200 patients with
a clinical diagnosis of FH who reside in the southeast of
England and in whom we intend to determine the
frequency of the previously described mutations in the
LDL receptor gene. In this article we describe the
identification of those patients in the group in whom the
mutant allele of the LDL receptor gene has a major
deletion or rearrangement that can be detected by
Southern blotting of genomic DNA with specific probes.
Methods
Selection of Patients
The majority of the patients in this study (189 of 200)
were selected at random from a group of patients with
a clinical diagnosis of heterozygous FH who have been
attending one of three lipid clinics in the London area
during the past few years (Hammersmith Hospital,
Charing Cross Hospital, and St. Mary's Hospital). The
diagnostic criteria for definite heterozygous FH in these
clinics require that the patient has a plasma cholesterol
concentration of 7.5 mmol/1 or above (6.7 mmol/1 if 16
years old or younger) and that detectable tendon xanthomas are present in the patient or a first- or seconddegree relative. If no tendon xanthomas are present, the
patient is given a diagnosis of possible FH if a hypercholesterolemic first-degree relative suffered a myocardial infarction before the age of 60 years (or 50 years in
a second-degree relative).8 The other 11 patients had
homozygous FH, based on a plasma cholesterol level of
>15 mmol/1, the presence of cutaneous and tendon
xanthomas, cardiovascular involvement before puberty,
and hypercholesterolemia in both parents. Clinical details of most of these homozygous individuals have been
described previously.9 Genotype analysis based on four
polymorphic sites in the LDL receptor gene showed that
at least seven of these clinically homozygous patients
were compound heterozygotes with two different mutant alleles (J. Webb and A.K. Soutar, unpublished
observations). Thus, the sample of patients comprised
not less than 207 defective alleles for the LDL receptor
gene.
All but one of any known related individuals were
excluded from the study, but no attempt was made to
select patients on the basis of cultural and ethnic origin.
Patients with a clinical diagnosis of FH who were known
to carry the gene for familial defective apolipoprotein
B3^0010 were excluded from this study.
Southern Blotting
Genomic DNA was isolated from frozen whole blood
(10 ml) or frozen packed blood cells by lysis of the cells
with Triton X-100 followed by phenol/chloroform extraction, essentially as described previously.11 The integrity and approximate concentration of the DNA were
determined by electrophoresis on 0.7% (wt/vol) agarose
gels stained with ethidium bromide; a known amount of
lambda phage DNA cut with Mndlll was included as a
764
Arteriosclerosis and Thrombosis
Vol 12, No 7 July 1992
FIGURE 2. Autoradiographs of
(a)PvuD digest + 1 9 1 3 probe
Southern blots of genomic DNA
from familial hypercholesterolemia (FH) patients to detect gross
deletions or rearrangements in the
low density lipoprotein (LDL) receptor gene. For Southern blotting, genomic DNA from FH patients (identified by a number
below each blot) or normal controls (N) was digested with either
(b) PvuII digest + 1 9 1 3 probe
-16.5Kb
-14Kb
—-16.5 Kb
-16.5Kb
-14Kb
Pvu // (panels a and b) or Bgl //
-3.5Kb
N 172
-3.5Kb
218 204 203 202 200
-3.5Kb
Normal controls
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(c) Bglll digest + 1700 probe
(d) Bglll digest + promoter probe
••» —-13Kb
i-lOKb
13Kb-»-
N 137 224 N
28 51 185 200 218 N
standard. For Southern blotting, 10 ng DNA was digested overnight at 37°C with 20 units of restriction
enzyme in 25 y\ of the appropriate buffer provided by
the supplier (Boehringer). The extent of digestion was
determined by electrophoresis of a portion of the digested DNA (l/25th of the volume) on an agarose
mini-gel, and the DNA was redigested overnight with an
additional 5 units of enzyme where necessary. The
digested DNA was then fractionated by electrophoresis
on a 0.7% (wtA'ol) agarose gel (13x18 cm) for 16 hours
at 45 V. The fractionated DNA was denatured with
alkali and transferred to a nylon membrane (Hybond-N,
Amersham International) by capillary blotting in 20 x
saline-sodium citrate buffer for a minimum of 18
hours.12 The DNA was fixed to the membrane by
exposure to UV light for 3 minutes on a transilluminator
(302-nm wavelength; UVP Inc., Genetic Research Instruments). The membrane was prehybridized for 1
hour and hybridized for 18 hours at 65°C in 0.5 M
sodium phosphate, pH 7.2, 7% (wtA'ol) sodium dodecyl
sulfate, 1% (wtA'ol) bovine serum albumin, and 1 mM
EDTA. 13 For hybridization, ^P-labeled DNA probes
were added at 2-5xlO 6 cpm/ml. Blots were washed
briefly three times with 0.0 4 M sodium phosphate, pH
7.2, 1% (wtA'ol) sodium dodecyl sulfate, and 1 mM
EDTA and then for 1 hour at 65°C. Autoradiography
was performed for 24 hours to 10 days at -70°C with
185 184 183 182 181
(panels c and d) and hybridized
with a nP-labeled probe as indicated. Expected restriction fragments (see Figure 1) are indicated
by arrows. Panel A: Southern blot
for three FH patients in whom
additional bands were detected on
the Pvu / / blot (FH172, 200, and
218); panel b: Southern blot for
normal controls who are homozygous ( + / + , —/—) and heterozygous (+/-)for the presence
( + ) or absence ( - ) of the Pvu / /
site in intron 15; panel c: Southern blot for FH patients in whom
additional bands were detected on
the Bgl / / blot; and panel ±
Southern blot for FH patients who
are heterozygous for an uncommon Bgl / / restriction fragment
length polymorphism in the 5'
flanking region of the LDL receptor gene.
Kodak X-Omat AR film. Blots were stripped for rehybridization by washing with 5 mM tris(hydroxymethyl)
aminomethane HC1, pH 7.5, at 75°C for 30 minutes.
DNA Probes
Two cDNA probes were prepared from plasmid
pLDLR3, kindly provided by Dr. D. Russell, Dallas,
Tex. The 1913 probe comprised a 1.9-kb BamHl fragment extending from the BamHl site in exon 10 to that
in exon 18. The 1700 probe comprised a 1.7-kb Hindlllj
Bgl II fragment extending from the Hindlll site in the
polylinker of the vector to the Bgl II site in exon 12
(Figure I). 14 Exon-specific probes and a 720-bp fragment comprising exon 1 and the 5' flanking sequences
containing the promoter region for the gene were
prepared by polymerase chain reaction (PCR)-dependent amplification as described below. All probes were
purified by electrophoresis on low-melting-point agarose (Nusieve, FMC Biochemicals) and labeled with
32
P-deoxycytidine triphosphate by random-primed
synthesis.16
Gene Amplification
Fragments of the LDL receptor gene were amplified
by PCR17 essentially as described previously18 with
oligonucleotide probes located in the introns adjacent
to the exon or pair of exons to be amplified.5 The 5'
Sun et al
(a)
LDL Receptor Gene Deletions in UK
765
Bglll/EcoRV digest
+ promoter probe
BglH digest + pmnotcr probe
13Kb10Kbi-i
1-2 1-3 1-4 1-5 n-i n-3 n-4 n-5
n-2m-i
1-11-211-1
t
t
FH 120
(b)
1 Q
FH 120
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o Lri a
1+-
-4.3 Kb
FHU8
m
a
n-2
2+ -
3+-
4--
5+-
FH118
n
1+-
2+-
m
r+FIGURE 3. Characterization of the Bgl IIpolymorphism in the low density Upoprotein receptor gene. Panel a: Southern blot of
genomic DNA from members of the families of FH 120 and FH 118. Genomic DNA was digested with Bgl / / or a combination
of Bgl / / and EcoftF as indicated and hybridized with the 720-bp probe to the promoter plus exon 1. Numbers below each lane
refer to individuals identified in the pedigrees below. Panel b: Pedigrees ofFH 120 and FH 118 showing inheritance of the Bgl / /
restriction fragment length polymorphism; "+" indicates the presence and "—" the absence ofthe cutting site. Those individuals
whose DNA was available for study are identified by a number below the symbol Shaded symbols represent individuals with a
cUnical diagnosis for heterozygous familial hypercholesterolemia (FH). Affected individuals in the family of FH 118 are
heterozygous for a 3-bp deletion in exon 4s; the mutation causing defective low density Upoprotein receptor function has not yet been
identified in FH 120. a, Males; o, females; /, deceased.
primer for the fragment containing the promoter region
extended from nucleotides -621 to -601, where nucleotide 1 is the A of the mRNA AUG initiator codon of
the gene,1* and the 3' primer was in the intron adjacent
to exon 1. The details of individual PCR incubations
designed to amplify across a deleted portion of the gene
are given in the legends to the figures.
Results
When genomic DNA from 200 apparently unrelated
individuals with a clinical diagnosis of heterozygous
(189) or homozygous (11) FH was analyzed by Southern
blotting with cDNA probes specific for the LDL receptor gene, nine patients were found with an abnormal
restriction fragment pattern characteristic of a major
gene deletion or rearrangement (Figure 2). In three
patients, digestion of genomic DNA with Pvu II followed by hybridization with a 1,913-bp cDNA probe
encompassing exons 11-18 revealed an extra band (Figure 2a); also shown are the patterns obtained for three
normolipemic individuals that demonstrate the common
Pvu II polymorphism in intron 15 of the LDL receptor
gene (Figure 2b). In one of these patients and in
another seven patients, the restriction fragment pattern
obtained after digestion of genomic DNA with Bgl II
and hybridization with a 1,700-bp cDNA probe extending from exon 1 to exon 11 of the LDL receptor gene
revealed extra bands (Figure 2c). The Bgl II blots were
also hybridized with a 720-bp probe derived from exon
1 and the adjacent 5' flanking region (Figure 2d). As
well as the expected band of 10 kb, a second band of
approximately 13 kb was observed in approximately
13% of the individuals; two individuals with only the
13-kb band were identified. The expected 10-kb band
was detected weakly when the blot was probed with the
1700 probe, but the new 13-kb band was obscured by the
presence of the 13-kb band comprising exons 4-11.
When DNA from individuals with the 13-kb band was
digested with a combination of EcoRV and Bgl II, in
each case a single band of approximately 4.3 kb was
observed (Figure 3a), suggesting that the Bgl II site at
the 5' end of the promoter region was polymorphic
(Figure 1).
Based on the observations that 112 individuals were
homozygous for the common allele characterized by the
10-kb Bgl II fragment encompassing the promoter and
exon 1, two were homozygous for the infrequent allele
characterized by the 13-kb fragment, 17 individuals
were heterozygous at this site, the calculated frequency
of the rare allele in this group of FH patients was 0.08,
and distribution of the alleles was in Hardy-Weinberg
equilibrium. Mendelian inheritance of the infrequent
allele was observed in two families, those of FH 118 and
FH 120 (Figure 3b). In the family of FH 120 the
infrequent allele cosegregated with hypercholesterolemia and a clinical diagnosis of heterozygous FH; in the
766
Arteriosclerosis and Thrombosis
Vol 12, No 7 July 1992
TABLE 1. Summary of Results of Southern Blotting to Define Extent of Gene Deletions
Enzyme digesrf
Probe
Predicted
bands* (kb)
FH 22/224/51
BglH
1700
9+13
FH 22/224/51
FH 22/224
Pvu II
Xp/i VXba I |
1913
1700
3.5 + 16.5/14
15 (Faint 9+6)
None
FH51
AJJTJ I/^fta I |
1700
15 (Faint 9+6)
19
FH 28/137
FH 28/137
FH28
BglU
Pvu II
£coRI|
1700
1913
Various
9+13
3.5 + 16.5/14
10
12.5
None
10.5
FH137
Be/ IIII
Exons 7 or 8
13
12.5
FH172
II
1913
3.5 + 14
9.5
21
8.5
10.5
FH patient*
Extra
bands§ (kb)
12.0
14
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FH172
FH172
FH172
Bgi II
EcoRV/Xba I||
Kpn I
1913
Exons 12-15
Exons 12-15
9+11
None
FH218
Bgin\\
1700
9.5 + 13
-23
FH 218
Kpn I
Exons 12-15
19+9
17
FH200
Bgl 111
9.5 + 13
>23
FH200
FH185
FH185
FH185
Kpnl
Bgl II
Pvu II
EcoKl
Exons 12-15
1700
1913
1700
19+9
9.5 + 13
3.5 + 14+16.5
10+1.7+8
-18
None
4+(12
FH185
FH 185
Sac VEcoKV
Kpn VXba I
1700
1700
15
None
15 (Faint 9+6)
19
170
°
5.5
Conclusions and comments
Extra band not detected by probes to exons 3, 4, and
5+6; detected by probe to exon 7.
Bgl II site at intron 11/exon 12 intact.
Xba I site in intron 1 intact; — 10-kb deletion of exons
2-6.
Deletion includes Xba I site in intron 1; probably
>10-kb deletion including promoter to intron 6.
Bgi II site at intron 11/exon 12 intact.
Detected by probes to exons 3 and 4 but not to exons
5+6 or 8; —1-kb deletion including EcoRl site in
exon 5. Confirmed by PCR across exons 4-6.
Extra band detected by probe to exon 8 but not by that
to exon 7; —1-kb deletion of exon 7.
Extra band not detected by probe to 5' end of exon 18,
but band suggests Pvu II site in exon 18 probably
intact.
—10.5-kb deletion between exons 12 and 18.
EcoKV site in intron 14 intact.
Kpn I site in intron 14 intact, but intron 15 missing.
Deletion extends from mid intron 14 to mid exon 18.
Detected by 1913, exons 4 and 5+6 but not exon 3.
Deletion of — 11-kb including Bgl II site at intron
11/exon 12.
Exon 14 missing, but exon 15 partially present.
Deletion extends from intron 6 to intron 14.
Detected by 1913||, exons 4 and 5+6 but not exon 3.
Deletion of —10-kb including Bgl II site at intron
11/exon 12.
Similar to FH 218, but deletion slightly smaller.
-17
Bgl II site at intron 11/exon 12 intact
12 kb detected by probes to exons 3, 4, and 5+6 but
not exon 8 (probably from partial cleavage of EcoKl
site in exon 5); 4 kb detected by probe to exon 8, but
not exons 3-6.
Gene normal between exons 2 and 6.
Duplication (4 kb) of exons 7+8 in intron 6. Extra
4-kb band on EcoRl blot from duplication of site in
intron 6.
'The number is an arbitrary identification assigned to the patient for this study.
tGenomic DNA from each individual was digested with the restriction enzyme indicated and hybridized with 32P-labeled probe as
described in "Methods."
tBands predicted from the restriction enzyme map of the low density lipoprotein receptor gene (sec Figure 1).
§Extra bands that were not detected on blots of DNA from unaffected individuals.
||Data shown in Figure 2 or 4.
FH, familial hypercholesterolemia; PCR, polymerase chain reaction.
family of FH 118 the infrequent allele cosegregated with
the occurrence of a 3 -bp deletion in exon 4 of the LDL
receptor gene that is known to cause defective LDL
receptor function.3 Apart from this polymorphism, no
unexpected bands were observed for any individual on
blots of Bgl H-digested DNA hybridized with the promoter probe.
To analyze the nature of the gene defect in each case,
further enzyme digestions of genomic DNA were performed and the blots hybridized with exon-specific
probes as described in Table 1. Southern blots showing
a characteristic pattern of bands for each deletion are
shown in Figure 4.
Discussion
Of the 200 FH patients in this sample from lipid
clinics in the London area, nine have been found to be
heterozygous for a defective LDL receptor gene in
which a major deletion or gene rearrangement was
detectable by Southern blotting. The data are summarized in Table 2. Because 11 of the 200 patients are
clinically homozygous and at least seven of these are
Sun et al
(a) KpnVXbal digest
+ 1700 probe
(b) Bgin digests
+exon 7 + exon 8
probe
probe
LDL Receptor Gene Deletions in UK
(c)Bgffl digests
+ 1913 probe
767
(d)XbaVEcoRV digest
+ exous 12-15 probe
15Kb•
13Kb
9Kb6Kb-
8.5Kb-
21Kb-*N 137
51
N
N 137
200 N 218 N
22
(e) EcoRI digests
+ 1700 probe
+ exon 3
probe
+ exon4
probe
+exon 5&6
probe
+ exon 8
probe
172 N
(0 PCR-ampliflcation of exoos 4-6
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:
28
N 185
28
N 185
28
N
185
28
N
185
28
2.4 Kb
1.4Kb
N 185
FIGURE 4. Southern blots confirming deletions and rearrangements in the low density lipoprotein receptor gene of familial
hypercholesterolemia (FH) patients. Panels a-e are autoradiographs of Southern blots of genomic DNA from FH patients
(indicated by the number below each blot) or normocholesterolemic controls (N) digested with restriction enzyme(s) and
hybridized with nP-labeled probes as indicated. Expected restriction fragments (see Figure 1) are indicated by arrows. Panel f:
Genomic DNA from FH 28 and 137 and a normal control amplified by polymerase chain reaction (PCR) with oligonucleotides
located at the 5' end ofexon 4 and the 3' end ofexon 3. In the PCR, denatwation was for 2 minutes at 95°C, and annealing and
extension were for 8 minutes at 65°C for a total of 40 cycles. Samples of the PCR mix (5 yl) were fractionated on 0.7% (wtfvol)
agarose gels stained with ethidium bromide. Size of DNA markers (in kilobases) is indicated on the left, and the sizes of the
amplified fragments obtained are on the right. MW, molecular weight.
compound heterozygotes with two different defective
alleles, the nine mutant alleles represent approximately
4.5% of the total. This value is similar to that found
when other groups of FH patients in the United Kingdom21 or Canada19 have been screened for major gene
deletions or rearrangements in this way. However, in a
Dutch population, 17.5% of 53 FH patients have recently been found to have a mutant allele that could be
detected by Southern blotting, but this rather high
frequency resulted from the presence of one common
deletion in more than half of these individuals.22
Only one of the deletions identified in our study was
present in more than one of the 200 unrelated individuals, but a number of them have been observed previously in FH patients of British or European ancestry.
For example, the gene defect identified in two patients
in this study (FH 22 and 224), one of whom is a
compound heterozygote, was a 10-kb deletion that
appears to be identical to that found in a Canadian
patient (identified as FH 49) characterized by Langlois
and coworkers.19 Of the six patients with deletions in
the LDL receptor gene identified by these authors, five
were apparently of English or Irish origin, and thus, it is
likely that all of the individuals with this deletion have
inherited the same mutant allele from a common ancestor. The phenotype of cells expressing this mutant allele
has not been determined, but even if a receptor protein
with such a large deleted fragment was synthesized and
processed, then it would at best be markedly defective
in its ability to bind LDL.
The deletion of 1 kb encompassing exon 5 that we
have observed in a single individual (FH 28) has also
been described previously in two patients of European
origin, one English21 and the other French.20 The phenotype of the LDL receptor protein in cells expressing
this mutant allele has been shown to be binding defective, with LDL binding being more severely affected
than binding of lipoproteins containing apolipoprotein
E.20 It is also probable that the 1-kb deletion of exon 7
that we have observed in a single patient (FH 137) is the
same as that described in a patient of English origin by
Horsthemke et al.21 Cultured cells are not available
from FH 137, but because the donor splice sites for
exons 6 and 7 are in frame,14 the resultant abnormal
mRNA should code for a receptor protein that has the
first growth factor-like repeat A in domain 2 missing but
is otherwise normal. By analogy with a similar mutation
introduced by site-directed mutagenesis and expressed
in heterologous cells, the phenotype of such a receptor
would be mildly binding defective.25
The other five defective LDL receptor genes identified by Southern blotting in this sample of FH patients
in the United Kingdom do not appear to have been
described previously, although there are similarities
768
Arteriosclerosis and Thrombosis
Vol 12, No 7 July 1992
TABLE 2. Summary of Deletions and Rearrangements
Plasma
cholesterol
(mmol/1)
Diagnosis
Ethnic origin
Mutation
Comments
FH22 l
10.0
Htz
UK
Approximately 10-kb deletion of
exons 2-6
Same as FH 49 described by Langlois
et al1'
FH224J
FH51t
26.0
16.3
Compound htz*
Compound htz$
UK
UK
Not previously described
FH28t
8.9
Htz
UK
FH 137f
8.7
Htz
UK
FH185
8.3
Htz
Polish
>10-kb deletion of promoter to
exon 6
Approximately 1-kb deletion of
exon 5; confirmed by PCR
Approximately 1-kb deletion of
exon 7
Duplication of exons 7 and 8 in
intron 6
FH200
8.3
Htz
Asian Indian
FH218
12.0
Htz
UK
FH172t
15.9
Compound htz
UK
FH
patient
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Approximately 11-kb deletion of
exons 7-14
Approximately 10-kb deletion of
exons 7-14
Approximately 10.5-kb deletion
of exons 15-18
Same as FH Paris20 and FH London
221
? Same as KU>
Not previously described, but common
deletion of exons 7 and 8 in Dutch
FH patients22
Not previously described
? Similar to FH Osaka 2P
Not previously described
Htz, heterozygote; compound htz, clinical diagnosis of homozygous familial hypercholesterolemia (FH) but two different mutant alleles;
PCR, polymerase chain reaction.
*The other mutant allele has not been characterized.
tAffected relatives with the same mutation available.
$The other mutant allele in this individual has a point mutation (Glua)-»Lys).24
with deletions found in FH patients elsewhere. For
example, the deletions in FH 200 and FH 218 in our
study that both encompass exons 7-14 are similar to a
deletion in a Japanese patient described by Miyake et
al.23 The deletion in this patient was believed to be
caused by misalignment of Alu sequences in intron 6
and intron 14, and although we have not cloned the
deletion joint in our patients, it is likely that the deletion
in FH 200 is identical to that in the Japanese patient,
while that in FH 218 involves different Alu repeats and
TABLE 3.
results in a slightly larger deletion. If the primary
transcripts of RNA are spliced to produce a receptor
protein that lacks domain 2 but is otherwise normal, as
has been observed in cells from the Japanese patient,23
then the mutant alleles in FH 200 and FH 218 will result
in the same recycling-impaired phenotype.
The large deletion at the 5' end of the gene that we
have observed in FH 51 is superficially similar to that
described recently in an Italian patient by Lelli et al26
(identified as FH 44 or FH Bologna). However, the 5'
Summary of Mutations in the LDL Receptor Gene in Familial Hypercbolesteroleniia in the United Kingdom
Type of mutation
Deletions/rearrangements detected by
Southern blotting
No. of
unrelated patients
9*
5t
Comments
4/9 Same as and 3/9 similar to
previously detected mutations
1/9 Insertion complementary to
previously described deletion
Recurrent mutation
Reference
This article
11
30
5*
Probably same allele
24
Unpubl obs
Exon 4 mutations: deletion of Gry197
New deletion of 2 bp in codons 206/207
New CyS2io-»stop
Ser1J6-»Leu
Total
6t
5
15
3
15
Same allele as FH Piscataway
Same allele
Same as FH Maine/Afrikaner 1
Same as FH Puerto Rico, but
recurrent mutation?
3
Unpubl obs
Unpubl obs
6
31
35
(17% of mutant alleles)
*One clinically homozygous patient is a compound heterozygote in whom one allele has a deletion and the other a
point mutation (Glugo—>Lys).
tOne of the patients is a compound heterozygote in whom the other mutant allele has not yet been characterized.
SPatients (t, three of six) also heterozygous for Bgl II restriction fragment length polymorphism in 5' flanking region.
LDL, low density lipoprotein; FH, familial hypercholesterolemia; unpubl obs, unpublished observations.
Sun et al LDL Receptor Gene Deletions in UK
Downloaded from http://atvb.ahajournals.org/ by guest on May 10, 2017
limit of the deletion must be different in our patient, as
we did not observe the additional band of 7.5 kb on the
EcoKL blot, and the abnormal band on the Bgl II blot
was smaller than that observed with FH Bologna; the
deletion in FH 51 may extend farther at the 5' end of
the gene and would undoubtedly result in a null
phenotype.
The remaining deletion observed in this sample of
200 patients, that of 10.5 kb encompassing exons 14-18
in FH 172, has apparently not been observed previously.
Several mutant LDL receptor genes have been described in which exons 16-18 are deleted, namely the
Finnish mutation,27 FH Osaka-2 (FH 781 in Lehrman et
al28), and FH Rochester (FH 274 in Lehrman et al29),
but the restriction fragment patterns obtained with FH
172 differ, and blotting with exon-specific probes confirmed that exon 15 is also deleted in this patient.
Although we have not cloned the deletion joint, it is
likely that misalignment of Alu sequences in intron 14
and exon 18 is responsible for this deletion. Preliminary
studies (V. Gudnason et al, unpublished observations)
have shown that the mutant allele with this deletion in
cultured cells from FH 172 produces a truncated receptor protein that is not processed to a mature form and
resembles the abnormal protein detected in cells with
the Lebanese mutation, where a stop codon has been
introduced into exon 14 as the result of a point mutation
in codon 660/
The gene defect in the final patient in this study (FH
185) was a duplication of exons 7 and 8 inserted in
intron 6, a defect that has not been described previously. However, it is of interest that a 4-kb deletion of
exons 7 and 8 has been observed as a frequent cause of
the disease in Dutch FH patients,22 and it is tempting to
speculate that this allele in FH 185 represents the
complementary chromosome that could be formed
when such a deletion occurs as a result of unequal
crossing over. The phenotype has not yet been determined for this mutation.
In addition to the mutations described above, a new
restriction fragment length polymorphism for Bgl II in
the 5' flanking region of the gene was detected as a
heterozygous trait in 13% of the patients. The frequency of the rare allele, characterized by a 13-kb Bgl II
fragment compared with 10 kb for the common allele,
was 0.08 in this group of FH patients. Although we have
not attempted to screen normal individuals for this
restriction fragment length polymorphism, it is unlikely
that it represents a common deleterious mutation responsible for FH in these patients, as it was found in
some heterozygous FH individuals in whom a point
mutation or a small deletion in the gene that is known to
cause defective LDL receptor function had also been
identified (Table 3).
In summary, of the six deletions found in patients of
apparently British ancestry in our sample of 200 patients, three have been identified previously in FH
patients of UK origin. The remaining three mutations
have not been described but are deletions in regions
that seem to be susceptible due to the presence of
numerous Alu sequences.3 The low frequency of each
individual deletion in the LDL receptor gene as a cause
of FH makes it difficult to predict whether the deletions
in this population of 200 FH patients are representative
of those that will be found in other FH patients in the
769
United Kingdom. This low frequency of each individual
deletion is a little surprising, as this same group of 200
patients has now been screened for a number of known
mutations in the LDL receptor gene (V. Gudnason et
al, unpublished observations, and References 24 and
30), and as summarized in Table 2, the majority of these
have been observed in more than one unrelated patient.
Indeed, several mutations, including Pro^-^Leu, 3 0
Glugo-»Lys,24 and the deletions of G l y ^ and of 2 bp at
the 3 end of exon 4 (V. Gudnason et al, unpublished
observations), are present in 2-3% of the patients. The
available data suggest that at least some of these more
frequently occurring mutant alleles have all been inherited from a single ancestor of English origin and that
this value of 2 - 3 % could represent the highest frequency that can be expected for any mutation causing
FH in the United Kingdom. However, the 200 patients
in this study have been drawn from the population
around London and the southeast of England and may
comprise a more heterogeneous group than would be
encountered elsewhere in the United Kingdom. Indeed,
one of the point mutations (Glugo—>Lys) has been found
at a much higher frequency in a sample of FH patients
attending a lipid clinic in Manchester, where the population may have been more stable over the last
century.24
Acknowledgments
The authors are grateful to Mrs. E. Manson for excellent
secretarial help; to Dr. D. Russell, Dallas, Tex., for plasmid
pLDLR3; and to Ms. S. McCarthy for invaluable information
about the families of some of the patients in this study. We are
grateful to Dr. J. Lloyd, Institute of Child Health, for providing
samples from FH 172 and his family.
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Characterization of deletions in the LDL receptor gene in patients with familial
hypercholesterolemia in the United Kingdom.
X M Sun, J C Webb, V Gudnason, S Humphries, M Seed, G R Thompson, B L Knight and A K
Soutar
Arterioscler Thromb Vasc Biol. 1992;12:762-770
doi: 10.1161/01.ATV.12.7.762
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