Clinical Chemistry 45: 1424-1430, 1999;
(Clinical Chemistry. 1999;45:1424-1430.)
© 1999 American Association for Clinical Chemistry, Inc.
Long-Distance PCR-based Screening for Large Rearrangements of the LDL Receptor Gene in Korean Patients with Familial Hypercholesterolemia
Sung Han Kim1,
Ji Hyun Bae2,
Jae Jin Chae2,
Un Kyung Kim2,
Seong-Joon Choe3,
Yong Namkoong4,
Hyo-Soo Kim3,
Young-Bae Park3 and
Chung Choo Lee1,a
1
Department of Biology and SRC for Cell Differentiation, and
2
Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea.
3
Department of Internal Medicine, Seoul National
University Hospital, Seoul 110-799, Korea.
4
Department of Biology, Kangnung National University,
Kangnung 210-702, Korea.
a Address correspondence to this author at: Department of Biology, College of Natural Science, Seoul National University, Seoul 151-742, Korea. Fax 82-02-872-1993; e-mail chunglee{at}plaza.snu.ac.kr
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Abstract
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Background: The LDL receptor is a cell-surface protein that
regulates plasma cholesterol by specific uptake of LDL particles from
the blood circulation. Familial hypercholesterolemia (FH) results from
defective catabolism of LDL, which is caused by mutations in the
LDL-receptor gene.
Methods: For the rapid and reliable detection of large
rearrangements in the LDL-receptor gene, we established a screening
method based on long-distance PCR as an alternative to
Southern-blot hybridization. Using long-distance PCR, 45 unrelated
Korean subjects heterozygous for FH were screened to assess the
frequency and nature of major structural rearrangements in the
LDL-receptor gene.
Results: Two different deletion mutations, FH6 (same type as FH3
and FH311) and FH 32, were detected in four families by long-distance
PCR. Detailed restriction mapping and sequence analysis showed that FH6
was a 5.71-kb deletion extending from intron 8 to intron 12 and that
FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence
analysis for the breakpoints of all deletions detected in Korean FH
patients showed that only the left arms of the Alu repetitive sequences
were involved in the deletion event.
Conclusions: The screening method based on long-distance PCR
provides a powerful strategy for the detection of large rearrangements
in the LDL-receptor gene and is a rapid and reliable screening
alternative to Southern-blot hybridization.
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Introduction
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Familial hypercholesterolemia (FH) is a common autosomal dominant
disorder caused by a defect in the LDL-receptor gene; the frequency of
FH heterozygotes is ~1 in 500 in most populations (1). FH
is characterized by a selective increase of plasma LDL-cholesterol,
giving rise to tendon and skin xanthomatosis and to premature coronary
heart disease (1)(2).
Mutations of the LDL-receptor gene are genetically heterogeneous, and a
large number of mutations (to date, >300) including both large and
small rearrangements have been reported in various populations and
ethnic groups (1)(3)(4)(5)(6)(7). These mutations, which
disrupt the synthesis, intracellular transport, ligand-binding
capacity, internalization, and recycling ability of the LDL-receptor
protein, have revealed many aspects of the structure-function
relationship of the LDL-receptor gene
(1)(8)(9).
The vast majority of the large rearrangements of the LDL-receptor gene
are deletions of various sizes along the entire length of the gene
(10)(11). Most rearrangements have arisen by
recombination between two Alu repeats oriented in the same direction,
and these account for relatively high percentages of the mutations in
the LDL-receptor gene for FH in some populations
(1)(7)(12)(13)(14)(15)(16).
In general, Southern-blot hybridization has been used in screening
large rearrangements of various genes, including the LDL-receptor gene.
However, standard PCR techniques can provide a rapid and reliable
screening alternative to Southern-blot hybridization for the detection
of various large rearrangements in the LDL-receptor gene in FH
patients. We therefore established a screening scheme for large
rearrangements of the LDL-receptor gene using long-distance PCR. We
screened 45 unrelated FH heterozygotes using this scheme and identified
two large deletion mutations in four FH families: one is a novel 2-kb
deletion including exon 7, and the other is a 5.7-kb deletion including
exons 9–12. Segregation analysis in all four families confirmed the
relationship between the described mutation and the FH phenotype.
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Materials and Methods
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subjects
FH patients were screened from lipid clinics at the Department of
Internal Medicine of Seoul National University Hospital in Korea. A
diagnosis of heterozygous FH was made based on the following criteria:
LDL-cholesterol >2.6 g/L, a positive family history of
hypercholesterolemia or early coronary artery disease, and
Achilles tendon xanthomas. Patients were required to meet at least two
of the above criteria to be included in this study.
biochemical analysis
Triglycerides, total cholesterol, and
HDL-cholesterol were analyzed by enzymatic methods, and
LDL-cholesterol was calculated using the Friedewald formula
(17)(18).
screening of large rearrangements of the ldl receptor
using long-distance pcr
Genomic DNA samples were prepared using peripheral blood from 45
unrelated patients heterozygous for FH and from their families, using a
method described elsewhere (19). A long-distance PCR-based
method was established to screen large rearrangements in the
LDL-receptor gene covering ~45 kb. Table 1
shows the primers used for long-distance PCR amplification in
this study. Approximately 400 ng of genomic DNA was amplified in
50-µL reactions containing 400 nmol/L each primer, 500 µmol/L each
dNTP, 2.25 mmol/L MgCl2, and 0.75 units of
ExpandTM 20 kbplus enzyme
mix (Boehringer Mannheim) according to the manufacturer's
instructions. The PCR reactions were performed in a Perkin-Elmer
GeneAmp PCR systems 480. Amplification was performed using a three-step
PCR under the following conditions: an initial denaturation period for
2 min at 94 °C, followed by 16 cycles of 30 s of annealing and
extension at 68 °C and then 14 identical cycles in which the
extension time was automatically prolonged by 15 s per cycle, and
a final 10-min extension step at 68 °C. The annealing temperatures
and extension times were 58 °C and 11 min for fragment 5, 60 °C
and 11 min for fragment 3, 63 °C and 11 min for fragments 2 and 4,
and 64 °C and 17 min for fragment 1.
restriction mapping of deletion breakpoints and sequence analysis
To determine the deleted portion including the deletion
breakpoints, exon 6 to exon 13 (fragment 2) was amplified from FH
probands (FH3, FH6, FH32, and FH311) and from a normolipidemic control.
These PCR products were purified and digested with AvaII
enzyme according to the manufacturer's instructions (Promega) and were
separated on 1.5% agarose gels by electrophoresis and visualized by
ethidium bromide staining. Fine mapping of the deleted
portion was performed with several restriction enzymes, using methods
similar to those of Chae et al. (20).
All restricted fragments encompassing the deletion breakpoint and
corresponding fragments from a wild-type allele were subcloned into
pBluescript® SK+/- vector. DNA sequencing was
performed on these recombinant DNA templates, using the
dideoxynucleotide chain-termination method (21) with the
enzyme Sequenase (United States Biochemical),
[
-35S]dATP as a labeled nucleotide and both
pT7Blue T-vector and pBluescript as specific oligonucleotide primers.
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Results
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long-distance pcr-based screening scheme
To identify large structural rearrangements of the LDL-receptor
gene in FH patients, we established a new screening method based on
long-distance PCR. Using the long-distance PCR method, we amplified
five overlapping fragments covering the whole LDL-receptor gene from
the promoter to exon 18, ~45-kb (Fig. 1
A). The promoter to exon 5 region (fragment 1, 16.8 kb) was
amplified using the forward primer of the promoter and the reverse
primer of exon 5. Using the same method, we amplified the regions
covering exons 6–13 (fragment 2, 12.6 kb), exons 15–18, including the
noncoding region of exon 18 (fragment 3, 11.5 kb), exons 2–10
(fragment 4, 13.7 kb), and exons 12–18, excluding the noncoding region
of exon 18 (fragment 5, 13.5 kb). Amplified fragments 1, 2, and 3
covered the whole LDL-receptor gene, and fragments 4 and 5 were used to
detect the rearrangements including the boundaries between fragments 1,
2, and 3.
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Figure 1. Screening schemes based on long-distance PCR.
(A), the 45-kb whole LDL-receptor gene could be
amplified in five fragments. Fragment 1 covered the
region from promoter to exon 5; fragment 2, exons 6–13;
fragment 3, exons 15–18 (coding region);
fragment 4, exons 2–10; fragment 5,
exons 12–18. (B), fragments 1–5 were amplified and run
in agarose gel by FIGE. Lane M, DNA size marker,
 -HindIII; lane 1, fragment 1;
lane 2, fragment 2; lane 3, fragment 3;
lane 4, fragment 4; lane 5, fragment 5.
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The different large-sized PCR products (fragments 1–5) amplified from
the human LDL-receptor gene could be discriminated by field inversion
gel electrophoresis (FIGE) in Seakem Gold agarose gels (Fig. 1B
).
Because FIGE could maximize the resolution of kilobase- to
megabase-sized DNA fragments in the desired region, running gels under
the appropriate FIGE conditions enabled us to detect size
differences of amplified PCR fragments by <1 kb.
In a previous report from our laboratory (20), we detected
two large deletion mutations by Southern-blot hybridization; FH29
(deletion of exons 7 and 8), and FH110 and FH33 (deletion of exons
9–12). To test the availability of long-distance PCR-based method, we
amplified fragments 2 and 4 from FH29 and fragment 2 from FH110 and
FH33 by long-distance PCR. All of the PCR products were heterozygous
for two bands, including a small band with a deletion.
Restriction and sequencing analysis of the deletion bands showed
results identical to our previous study (data not shown)
(20). Thus, we confirmed that the long-distance PCR can
provide a method that is suitable in sensitivity and rate of detection
as an alternative to Southern-blot hybridization.
screening of the ldl-receptor gene by long-distance pcr
After the genomic DNA of 45 FH heterozygotes was screened, we
found that four probands had large structural rearrangements of the
LDL-receptor gene. Table 2
describes the lipid profiles of these four FH heterozygotes and
their families. These four patients all displayed symptoms of early
atherosclerosis and had physical signs of FH, including arcus cornealis
and tendon xanthomas.
Long-distance PCR-based screening for exons 6–13 (fragment 2) from the
genomic DNA of 45 unrelated FH heterozygote revealed heterozygous band
patterns in four FH probands (FH3, FH6, FH32, and FH311). In addition
to the 12.6-kb wild-type fragment, a 6.89-kb mutant band was detected
in FH3, FH6, and FH311, and a 10.7-kb mutant band was detected in FH32.
To confirm that these mutant bands arose from the deletion of the
LDL-receptor gene in FH patients, we amplified the region encompassing
exons 6–13 from each family member and examined the cosegregation of
these mutant bands and the FH phenotypes for the affected family
members (Fig. 2
). Fig. 2
shows that members of the FH6 family with FH had a
6.89-kb abnormal band (the same as FH3 and FH311) and that in the FH32
family, a 10.7-kb band was cosegregated with family members with FH.
Except for the fragment 2 region, PCR products of all other regions
(fragments 1, 3, 4, and 5) showed no different-sized mutant bands
compared with the wild type.
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Figure 2. Long-distance PCR-based screening of fragment 2 (exons
6–13).
(A), long-distance PCR for fragment 2 of the FH6 family.
Lane M, DNA size marker, -HindIII;
lane C, control; lane I-1, proband;
lanes II-1 and II-2, sons of proband;
lanes I-2 to I-6, sisters and brothers of
proband; lane II-3, niece of proband.
(B), long-distance PCR for fragment 2 of the FH32
family. Lane M, DNA size marker,
-HindIII; lane C, control; lane
I-1, proband; lanes II-1 and
II-2, daughter and son of proband; lanes
I-2 and I-3, sister and brother of
proband.  and  ,
heterozygous;  and  , homozygous normal.
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mapping and sequence analysis of the deleted breakpoints
To define the deleted portions including the deletion breakpoint,
we performed AvaII restriction mapping. As shown in Fig. 3
, the region encompassing exons 6–13 has five restriction sites
for AvaII, and digestion with AvaII produced six
appropriately sized fragments useful for restriction analysis
(11). The AvaII restriction map of the region
encompassing exons 6–13 for FH6 showed that exons 9–12 were deleted
and that the 1.38- and 3.2-kb fragments hybridized to form a 3.0-kb
fragment (Fig. 3A
). In FH32, exon 7 was deleted, and the 2.3-kb band of
FH32 was, we believe, formed by recombination between the 2.81- and
1.13-kb fragments (Fig. 3B
).
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Figure 3. AvaII restriction mapping of FH6 and FH32.
(A), partial AvaII restriction map for
fragment 2 of FH6. (B), partial AvaII
restriction map for fragment 2 of FH32. The sizes of the restriction
fragments (kb) are indicated in the thin lines, which
correspond to AvaII restriction sites.
(C), PCR products of fragment 2 for control, FH6, and
FH32 were digested with AvaII and electrophoresed on a
1.5% agarose gel. Lane M, size marker,
-HindIII; lane C, wild-type fragment 2
digested with AvaII; lane 1, fragment 2
of FH6 digested with AvaII; lane 2,
fragment 2 of FH32 digested with Ava II. Up- and
downstream boundaries of the deletion are indicated by vertical
dotted lines on the map.
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The sequence comparison analysis of FH6 and FH32 is shown in Fig. 4
. Both DNA sequences corresponded to the left arm of an Alu
repetitive sequence. The nucleotide sequence of FH6 was perfectly
matched with normal intron 8 in the 5' region and with normal intron 12
in the 3' region. Thus, the breakpoint was within a 27-bp region
between bases -123 and -149. Sequence analysis for FH32 showed that
the breakpoint was within a 29-bp region between bases -38 and -66.
We have numbered the nucleotides according to the consensus numbering
system for Alu sequences (22).
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Figure 4. Nucleotide sequences across the deletion breakpoints in
FH6, FH32, and the corresponding regions of the wild-type LDL-receptor
gene.
(A), alignments of the sequences of FH6: sequence
I8, the wild-type sequence of intron 8; sequence
FH6, the sequence across the deletion breakpoint in the FH6
allele; sequence I12, the wild-type sequence of intron
12. Vertical lines left of position -123 and right of
-149 designate the limits of the deletion breakpoint.
(B), alignments of the sequences of FH32:
sequence I6, the wild-type sequence of intron 6;
sequence FH32, the sequence across the deletion
breakpoint in the FH32 allele; sequence I7, the
wild-type sequence of intron 12. Vertical lines left of
position -38 and right of -66 designate the limits of the deletion
breakpoint. Dots between sequences indicate positions at
which the sequences are identical. The sequences are numbered in
accordance with the Alu consensus sequence described by Deininger et
al. (22).
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Discussion
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To date, >30 different large deletion mutations of the
LDL-receptor gene have been described, and most of them were detected
by Southern-blot hybridization. However, Southern-blot hybridization is
a relatively time-consuming method and requires labor-intensive
procedures. Recently, several studies have used long-distance PCR
instead of Southern-blot hybridization to identify large deletion
mutations of the LDL-receptor gene (23)(24), but
their screening schemes were not systematic and did not cover the whole
LDL-receptor gene. Thus, to screen the large deletion mutations of the
LDL-receptor gene more systematically, efficiently, and accurately, we
established a new scheme based on long-distance PCR. This scheme was
based on the location of several Alu sequences that have been involved
in large rearrangements in several populations
(4)(11)(20)(23)(24)(25)(26).
Because the whole 45-kb LDL-receptor gene could not be amplified at
once, considering the location of Alu sequences, we designed an
amplification scheme for the LDL-receptor gene by dividing it into
three appropriate fragments (Fig. 1
, fragments 1, 2, and 3). To detect
the deletions involving the boundary regions between fragments 1 and 2
or 2 and 3, we designed two additional overlapping fragments (fragments
4 and 5).
Using this scheme, we screened 45 unrelated Korean FH heterozygotes to
detect large deletion mutations of the LDL-receptor gene. Two different
large deletions were detected in four families by long-distance PCR and
precisely characterized by restriction mapping and sequencing. Of the
two deletions, FH6 was identical to FH110, which was detected in our
previous study and had a 5.71-kb deletion extending from intron 8 to
intron 12 (20), whereas FH32 was a novel deletion extending
from intron 6 to intron 7.
In FH6, the translation reading frame was shifted and a truncated
protein, which terminates 83 bp after exon 13, was produced. It may be
classified as a null allele. In FH32, the translation reading frame was
preserved, but the cysteine-rich A repeat in the EGF precursor homology
domain was deleted, which is similar to FH Leuven-1 and FH Cape Town-2
(1)(27)(28). Thus, the defective LDL
receptor of FH32 may be transported to the cell membrane but may not
interact with LDL as a rule.
Sequence analysis revealed that both deletions (FH32 and FH6) occurred
between two Alu repetitive sequences that are oriented in the same
direction as in most of the large structural rearrangements within the
LDL-receptor gene described to date. Thus, we could hypothesize that
homologous recombination by unequal crossing-over during meiosis
produced the large deletions in FH6 and FH32. The deletion of FH32
involves the left arms in introns 6 and 7. The deletion of FH6 involves
the left arms of the Alu repetitive sequences in introns 8 and 12. FH6
has the breakpoint within the same 27-bp fragment
(5'-GCCTTCCCAAAGTGCTGGGATTACAGGC-3') as well as having the same
deletion of exons 9–12 present in FH110 and FH33 and detected
previously in our laboratory (20). These findings raise the
possibility that the left arm of the Alu repetitive sequence might have
more intrinsic instability than the right arm and that the specific
27-bp fragment within the left arm of the Alu repetitive sequence might
be an important factors in large recombinational rearrangement.
Including the two families FH110 and FH33 (20), to date we
have detected the same deletion of exons 9–12 in five families (FH3,
FH6, FH311, FH33, and FH110). This mutation accounted for 71.4% of the
large deletions detected and thus may be the most common deletion type
in Korean FH patients. To examine the existence of a unique inheritance
pattern in these five families with deletion of exons 9–12, we
performed haplotype analysis for six known restriction fragment length
polymorphism sites (TaqI, AvaII,
PvuII, MspIA, MspIB, and
NcoI sites) (29)(30)(31)(32)(33)(34). Haplotype analysis showed
that the haplotypes of the mutant alleles were not exactly identical
with one another (data not shown). The AvaII site (exon 13),
which is adjacent to the deleted region (exons 9–12), had
negative type in all five families. In addition, except for the
TaqI site, the AvaII, PvuII,
MspIA, MspIB, and NcoI sites were
identical (- - - - +) in the four families except for FH311.
Therefore, it is possible that the mutant alleles of the four families
may have originated from the same ancestor allele and the different
restriction fragment length polymorphism types of TaqI site
might have resulted from a recombination between the original mutant
allele and the other allele (35).
In conclusion, we propose that the deletion mutation of exons 9–12
of the LDL-receptor gene is the most frequent deletion type in Korean
FH patients and that the fragment 2 region may be used as the primary
screening target to detect for large rearrangements of the LDL-receptor
gene in Korean FH patients because all large deletions have been
detected in fragment 2. We believe that the defects of the LDL-receptor
gene of the remaining 41 patients are small structural rearrangements
or point mutations that could not be detected by long-distance PCR.
Further studies are required to identify these mutations, and these
studies are in progress. The screening method based on long-distance
PCR can provide a powerful strategy for the detection of large
rearrangements in the LDL-receptor gene. The availability of the
long-distance PCR assay as an alternative to Southern-blot
hybridization enables us to screen rapidly and reliably for
various large rearrangements and to detect deletions differing by <1
kb. Furthermore, long-distance PCR may be applicable to detecting
large rearrangements in other genes (e.g., Becker/ Duchenne muscular
dystrophy) or in analyzing gene structure and long repetitive sequences
(e.g., fragile X syndrome).
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Acknowledgments
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This work was supported in part by research grants from
the Seoul National University Hospital Research Fund and from the
Korean Sciences and Engineering Foundation through the Research Center
for Cell Differentiation at Seoul National University.
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Abstract
Introduction
