Clinical Chemistry 43: 427-435, 1997;
(Clinical Chemistry. 1997;43:427-435.)
© 1997 American Association for Clinical Chemistry, Inc.
Single-strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercholesterolemia
Steve E. Humphriesa,
Vilmundur Gudnason,
Ros Whittall,
Ian N. M. Day and
for the International Federation of Clinical Chemistry Scientific Divison: Committee on Molecular Biology Techniques
Centre for Genetics of Cardiovascular Disorders, University College of London Medical School, Department of Medicine, The Rayne Institute, 5 University St., London WC1E 6JJ, UK.
a Author for correspondence. Fax + 44 171 209 6212, e-mail s.humphries{at}medicine.ucl.ac.uk
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Abstract
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The identification of the specific mutation causing an inherited disease
in a patient is the framework for the development of a rationale for
therapy and of DNA-based tests for screening relatives. We present here
a review of the single-strand conformational polymorphism (SSCP)
method, which allows DNA fragments that have been amplified with
specific primers and PCR to be scanned rapidly for any sequence
variation. The general principles of the method are described, as are
the major factors that must be considered in developing an optimal SSCP
strategy, namely the length of the PCR fragment and the temperature of
the gel run. Options for sample denaturing gel characteristics and
detection of DNA fragments are discussed. In addition, several
modifications are presented that have been developed for
high-throughput mutational analysis. The application of these
techniques to screen for mutations in the LDL receptor gene in patients
with familial hypercholesterolemia are described.
Key Words: indexing terms: microtiter array diagonal gel electrophoresis • polymerase chain reaction • LDL receptor gene • inherited disease
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Introduction
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The purpose of this report is to illustrate the use of the
single-strand conformational polymorphism (SSCP) technique for mutation
screening in the LDL receptor gene and to discuss several adaptations
of published methods that improve throughput and that we believe are
appropriate for a disorder such as familial hypercholesterolemia
(FH).1
In the next few years such techniques will be
helpful in tackling molecular diagnosis and family tracing in the large
number of multifactorial disorders where gene–environment interaction,
low penetrance, late onset of symptoms, and genetic heterogeneity mean
that high-throughput approaches for mutation screening are required for
the development of cost-effective genetic tests. Several methods have
been published that allow rapid comparison of the sequence of specific
fragments of DNA amplified by PCR from different individuals
(1)(2), and we have found SSCP analysis to be
the most useful.
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General Principles of the SSCP Technique
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The SSCP technique (3) is a method capable of
identifying most sequence variations in a single strand of DNA,
typically between 150 and 250 nucleotides in length. Under
nondenaturing conditions a single strand of DNA will adopt a
conformation (presumably dependent on internal base-pairing between
short segments by foldback) that is uniquely dependent on its sequence
composition. This conformation will usually be different if even a
single base is changed. Most conformations seem to alter the physical
configuration or size sufficiently that, even though the variant
sequence has the same charge, the configuration-to-charge
(size-to-charge) ratio is different enough to be detectable as a
mobility difference upon electrophoresis through a retarding matrix
such as acrylamide gel. Typically, the duplexes will be from the same
PCR reaction for samples with possible genotypic differences. In many
laboratories [
-32P]dCTP is incorporated during the
PCR, and diluted PCR product is denatured by a brief boiling step,
after which the sample is loaded on a nondenaturing "sequencing"
(40 cm) acrylamide gel. The samples thus adopt a single-stranded
secondary structure because of the formation of interstrand base
pairing. However, although the samples have been diluted, they
concentrate upon entry into the gel, and this allows some reannealing
to take place. The considerations implicit in classical "Cot"
studies of reannealing (concentration of each single strand, DNA
complexity, and time) apply (4). Thus, to reduce the
proportions of PCR product that reform as a double strand and reduce
the amount available as single strand for polymorphism analysis, the
sample needs to be relatively dilute. When using thin gels with small
capacity for sample volume, this concentration consideration may limit
the choice of DNA detection methods, with radioisotope detection being
the method of choice because it is considerably more sensitive than
ethidium bromide or even silver-staining methods.
The "classical" SSCP protocol thus includes 32P in
the PCR mix for maximal detection sensitivity in the diluted sample,
and a gel with a long track length for maximal resolution of small
mobility differences in the DNA fragments (see Fig. 1
). However, in the interests of higher efficiency of detection,
greater convenience, or safety, several studies have been made with
other protocols. Restriction digestion of a large PCR fragment before
SSCP has been described (5). Multiplex PCR is also
possible, and SSCP can be combined with allele-specific PCR
(6)(7) to select alleles in complex sets
(e.g., HLA genes), or with dideoxy chain termination to localize the
approximate position of an SSCP variation in a sequence
(8).
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Figure 1. SSCP of the 3' end of exon 4 of the LDL
receptor gene.
Pattern of fragments seen from six disease-causing and one neutral
(intron) mutation (C- = T + 24). The samples shown here are all from
heterozygous patients, so all samples have normal and variant bands.
The direction of electrophoresis is towards the +, which denotes the
anode. SS denotes the single strands; the slowest migrating
band is representative of one single strand, with the lowest
band representing the other single strand (faint). The
middle bands are "shadows" that are possible PCR
artifacts. The different pattern of fragments that results from
different mutations in the same PCR product can be seen.
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In SSCP analysis, heteroduplex bands are often seen on the gel as a
useful byproduct of the procedure (9). Reannealing to form
double-stranded DNA has been discussed above, and when higher
concentrations of single-stranded DNA are loaded, e.g., for nonisotopic
detection methods, the double-stranded band is the predominant band.
For human DNA (except for the sex chromosomes), a PCR product usually
represents amplification from two alleles, one of each autosome of a
pair. When a sequence variation is present in the heterozygous state,
the classical SSCP picture would be of four single-stranded bands, the
sense and antisense strands of the "normal" allele, and the altered
mobility sense and antisense strands of the "variant" allele. In
addition, reannealing to double strands permits four possible products,
"normal" double strand, "variant" double strand, and two
heteroduplexes ("normal" sense/"variant" antisense; and
"normal" antisense/"variant" sense). The heteroduplexes have a
mismatch "bubble" that often alters their mobilities relative to
homoduplex, resulting in additional bands (see Fig. 1
). The combined
efficiency of SSCP plus heteroduplex analysis for detection of sequence
variations should be well in excess of the 80% estimated for each
individually (2).
Since RNA base pairing is more stable than RNA-DNA base pairing, RNA
might be expected to adopt a more conformational structure and hence be
more sensitive to sequence changes. Published evidence suggests that
this is the case (10) with detection of up to 95% of
variations, but the inconvenience of making RNA strands (which involves
the complexity and expense of introducing RNA polymerase promoters and
extra reaction steps) for SSCP has precluded widespread use. For FH, a
suitable source of RNA would be from lymphocytes, but success is
dependent upon equal representation of mRNA concentrations from the
normal and mutant allele, and with premature stop codons or splice-site
mutations this may not be the case (V. Gudnason, unpublished).
In our experience there are four important variables that must be
considered in designing an optimal SSCP strategy. These are the length
of the PCR fragment, the effect of temperature of the gel run, the
method of PCR denaturation, and the characterization of the gel. These
will be examined in turn.
single-strand length
The optimal length of a single strand seems to be ~150–200
nucleotides (11). In this size range, 70–90% of single
base substitutions are apparent on SSCPs. Presumably longer strands
exhibit relatively less conformational change by a single base
substitution, and shorter strands adopt less conformation in the first
place. On average, the reverse complement of a particular four-base
sequence will occur once every 4 x 4 x 4 x 4 =
256 nucleotides; thus it would be expected that a PCR single strand
would contain quite a few possible sequences to form four-base
double-stranded stems, but few significantly longer stems form
perfect-match duplexes. Such segments would be expected to melt and
hence lose any sequence-specific conformation below the melting
temperatures at which 10–15-mer oligonucleotides dissociate from their
target (12). The behavior of stem–loop folds would also
be expected to depend on guanosine plus cytosine content, since the
three-hydrogen-bond base pair, GC, is more heat-stable than the
two-hydrogen-bond base pair AT. We have observed for exon 4 mutations
in the LDL receptor gene that mobility shifts are more frequently
apparent in the upper strand, and one explanation for this might be
that the faster-migrating single strand, which is assumed to be more
compact, has sufficient stability so as not to be much affected by a
base change at the temperatures used. A better understanding of these
factors would be of value to be able to make predictive analysis of
specific sequences to maximize the mutation-detection efficiency of
SSCP in a fashion similar to the preplanning of denaturing gradient gel
electrophoresis experiments (13).
effect of temperature
Several studies have reported the use of different temperatures
for running SSCP, and typical conditions are either at room
temperature, or 4 °C with 50 or 100 mL/L glycerol. As discussed
above, the small regions of base pairing that are responsible for the
conformation of the single strands and thus the potential polymorphisms
are likely to have a melting temperature at or below the UK average
room temperature, and our experience is that during hot weather the
migration of bands changes considerably such that certain SSCPs are no
longer detected. The use of 4 °C standardizes these conditions, but
although we have not systematically investigated the effect of
temperature control on band sharpness or detection rate of SSCPs, we
and others have not found many additional SSCPs in samples run at other
than our standard conditions (14)(15). Gels
are routinely run at 5V/cm overnight at "room" temperature in an
air-conditioned room, which is typically ~22 °C, with minima and
maxima at 20 °C and 25 °C respectively. At this voltage, power
per gel is ~4 W, and gel warming is insignificant. A more expensive
option would be the use of a water-jacketed gel plate with a
circulating water bath.
pcr product denaturation
For convenience, PCR products are used without purification, but
spurious bands may result if the number of cycles is excessive or if
there is excessive residual PCR primer that may anneal to single
strands (16). By using high detection sensitivity for DNA
(i.e., 32P), the number of PCR reaction cycles can be
reduced (e.g., to 20) and the sample can be diluted 10–30-fold, which
will minimize annealing between single strands or between single
strands and PCR oligonucleotides. However, if less sensitive detection
methods are used (see below), less dilution is possible; stronger
denaturants added to the sample may help. Formamide, sodium hydroxide,
urea, and methylmercuric hydroxide (17) have been used.
Although toxic and requiring a fume hood, methylmercuric hydroxide
appears to be the most effective. Most protocols involve heating the
sample, immediate chilling on ice, then loading onto an apparatus
between 4 °C and 25 °C. A top layer of gel with formamide
incorporated has also been proposed to aid sample denaturation
(18), but this does not avoid the strand reannealing that
will occur when the single strands first enter the nondenaturing gel.
characteristics of the gel
Acrylamide is the commonly used matrix for DNA fragments in the
SSCP size range. The ratio of acrylamide to bisacrylamide cross-linker,
and the total acrylamide percentage, determine the sieving properties
of the gel. The buffer conductivity and concentration also influence
SSCP mobility, as do gel temperature and other additives such as
glycerol. Several publications have detailed the different effects that
these different conditions can have on resolving a particular sequence
variation. Reduced cross-linker ratio (bis:acrylamide 1:49) and 50–100
mL/L glycerol are popular (19), although other protocols
such as high-percentage gels can be useful (20). We have
found (Gudnason, unpublished) that the latter is true for a 340-bp PCR
fragment representing the 5' end of exon 4 of the LDL receptor gene.
However, there is no adequate theoretical basis to explain the
substantial influence of particular conditions in resolving certain
SSCPs, although some studies of folding and single-strand mobility have
been performed under nondenaturing and denaturing conditions (see
references in refs. 21 and 22). Hydrolink is an
acrylamide-like matrix polymerized by
N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate,
which is reported to have a more uniform pore size and is claimed to
give narrower bands and hence better resolution than acrylamide
(9).
Reported gel lengths range between 5 and 50 cm. At present, most
results are read by eye and therefore visible resolution is necessary.
Although clear-cut mobility shifts (e.g., 10%) are demonstrable on
short gels, a long electrophoresis may be necessary to resolve 0.5%
mobility difference. Long electrophoresis has the disadvantages of
needing a large apparatus, a higher voltage power pack, and more
complex arrangements to set up and control temperature. A longer run
broadens a band in accord with basic theory (effects of an imperfect
matrix and diffusion), and this may be compounded for SSCP without
proper temperature control (i.e., nonuniform conformation and hence
nonuniform mobility). Nevertheless, when it is important to avoid
false-negative results, long track length is advisable.
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Detection Methods for SSCP Single Strands
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Autoradiography of dried gels for 32P
incorporated during PCR as [-32P]dNTP (or as
[
-32P]ATP end-labeling of primers or of PCR product)
involves the hazards and inconvenience of radioisotope usage. The main
options that have been explored are silver staining
(23)(24) and ethidium bromide fluorescence
(18). Ethidium bromide intercalates with 5–10-fold higher
affinity in double-stranded DNA, and is therefore not well suited to
single strand detection. Nevertheless, ethidium bromide staining is a
one-step process, and conditions to load sufficient single-stranded DNA
have been achieved (17)(18). Sensitive
silver-staining protocols are available for DNA, with detection down to
1–10 pg/mm2 (25). The catalytic process of
silver reduction is initiated on DNA bases. A typical protocol involves
deposition of silver nitrate on bands, then reduction of silver by
formaldehyde and sodium carbonate to give a brown color. The reaction
is stopped by acetic acid.
Large gels (e.g., 30 x 40 x 0.04 cm) are difficult to
handle for postelectrophoretic staining. Binding the gel to one glass
plate with -methacryloxypropyltrimethoxysilane is done for silver
staining, but this renders gel recovery (as a dried image on Whatman
3-mm paper) very difficult. A photographic film-based process can take
a direct imprint from the gel while simultaneously amplifying the
signal (Promega, Southampton, UK). We find that our staining protocol
can be used, with complete adherence of the gel to a glass plate, by
using 4 µL/cm2 of 0.5%
-methacryloxypropyltrimethoxysilane:0.5% glacial acetic
acid:ethanol, and that the gel can then be reliably recovered onto
Whatman 3-mm paper (26). The single-strand band intensity
depends on PCR fragment sequence and size (some reanneal more readily
than others) and tends to be faint.
A further possibility not yet fully explored is to blot the gel, most
conveniently by "direct blotting." DNA is electrophoresed off the
end of the gel onto a revolving blot (27), and then one of
several methods of detection by hybridization can be used.
Automated DNA sequencers perform electrophoresis and detection on gels
similar to manual sequencing gels. However, the detection system is
usually fluorescence, either with oligonucleotides with fluorescent
labels attached, or incorporating fluorescent nucleotides during
polymerase reactions. Such apparatus can also be applied to SSCP by
using fluorescent labels (28). The arguments pertinent to
throughput, sensitivity of detection, and resolution are similar to
those for automated sequencing (29). The main shortfalls
are that one sequencer can only run one gel at a time, that there is
high capital expenditure, that access to "primary data" is
impossible if computer corrections for differential effect of different
dyes on mobility are involved, and that there is the need for
additional sophisticated workstations for secondary editing and
interpretation of the data.
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Rapid Throughput Modifications for Sample Handling
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We have made several major improvements in the techniques used for
sample handling that allow SSCP or other such mutation analyses to be
carried out in an extremely rapid manner (30). Tube
handling and labeling time has been reduced by storing DNA from
individual patients in a 96-well format. These arrays can easily be
"replicated" 20–30 times in 96-well microtiter plates for
subsequent PCR amplification and mutation screening or other
genotyping. The use of 96-well microtiter arrays and compatible
multichannel devices is well established for cell culture and for
analytical reactions. There is no labeling of tubes and the plate is
also the storage rack (with identity of sample being related to its
position in the array). The storage of many such plates, each
containing a small volume of pre-PCR template, in refrigerators or
freezers is inconvenient and expensive. To overcome this problem, we
allow the DNA templates to dry overnight at room temperature and store
all such plates at room temperature. Additional advantages are that
because the template DNA is dry, it has no volume to be taken into
account when setting up the PCR reaction and it also reduces the
probability of cross-contamination when using an automated multipipette
to aliquot the PCR mix. Several wells are left empty of DNA in each
array, so that cross-contamination can be monitored and control samples
can be added when necessary. In several months of using this procedure,
we have not found any cross-contamination. For samples from which
repeated and different PCRs will be undertaken, this enormously reduces
necessary staff time and any requirement for laboratory equipment for
storage. We routinely adjust the DNA concentration in the master array
to an average of 16 ng/µL water, so that obtaining 40 ng of dried DNA
requires repeated pipetting of 2.5-µL aliquots with multichannel
pipettes; it is much easier to pipette repeatedly 2.5 µL rather than
1 µL of a more concentrated stock with standard tips, and 2.5 µL is
a small enough volume to dry in a few hours on standing at room
temperature. Twenty replicas from one master array, consuming 96 tips
in total rather than 20 x 96 tips, can be prepared with an
eight-channel pipette by one worker in 2 h. Storage of pre-PCR
dried plates at room temperature makes it possible to prepare many
identical replicas in advance, when many different PCRs are planned
from a given master array, whereas advance preparation of replicas
would not be possible if there were a requirement for refrigeration or
freezing.
The setup of these PCRs is extremely simple, since the sample has zero
volume, so that unless the exact quantity of template DNA is critical,
any volume of a PCR master mix containing all components except
template DNA can be added to the well. The imprecision introduced by
pipetting is also minimized. The making and distributing of a PCR mix
and oil to all wells of a dried plate with a repeating dispenser takes
one worker ~10 min. We have also recently described, for PCR checking
and other analyses, a system for preparing and stacking open-faced
horizontal polyacrylamide gels, in which the wells retain the 96-well
array [microtiter array diagonal gel electrophoresis (MADGE)]
(31).
We have also modified the gel system to allow a 5–10-fold higher
throughput, and these modifications have been described in detail
(30). Briefly, two gels are poured between one large glass
sequencing plate (33 x 42 cm) and two outer smaller plates
(33 x 39 cm), so that two gels can be subjected to
electrophoresis simultaneously on one apparatus. Second, after first
loading the gel, the samples are electrophoresed at 400 V until the
bromphenol blue has run 2.5–3 cm, at which point the electrophoresis
is stopped and a second set of SSCP samples is loaded in the same way
as the first samples. Before loading the second time, the sharktooth
comb is removed and repositioned half a well across from its original
position. This enables the first and second loading bands to be
distinguished more easily. The first and second loading single and
double strands need to be nonoverlapping if the gel is to be
informative. The calculation of the timing of the second loading is
possible if the relative mobilities of the two single strands, one
double strand, and marker dyes are already known for the set of
conditions to be used. These mobilities were determined in a prior
experiment with single loading. For repetitive loading to be applied to
both the same and different exons, we prepared a computer simulation
that allows the solution of equations relating relative mobilities for
boundary conditions, including timing of loading, range of migration
distance desired, and time delays between loadings (available from G.P.
Weavind, 73064,3063@compuserve.com by e-mail request). Double
loading the gel means that a whole 96-well microtiter array of samples
may be analyzed on one gel. Finally, the sharktooth combs used are
custom cut with 4.5-mm spacing tooth-to-tooth to allow the use of a
multichannel pipette compatible with microtiter plates (9-mm
well-to-well spacing) for loading the SSCP gels. This results in every
second well being loaded with a multichannel pipette so that two
adjacent columns or rows of a microtiter plate are interleaved (see
Fig. 2
).
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Figure 2. Autoradiograph of a double-loaded SSCP gel of
exon 3 of the LDL receptor gene.
Exon 3 was amplified by PCR with primers flanking exon 3. The sequence
of the 5' primer is 5'-TGACACTTCAATCCTGTCTCTTCTG and for the 3'
oligonucleotide 5'-ATAGCAAAGGCAGGGCCACACTTAC, to give a product of 172
bp (4). Oligonucleotides were obtained from Genosys (The
Woodlands, TX). The amplifications were performed in an automated
thermal cycler (Hybaid Omnigene, Cambridge, UK) with Taq DNA polymerase
(Gibco BRL, Paisley, UK) in a total volume of 20 µL and overlaid with
20 µL of paraffin oil, under previously described cycling conditions
(30). The fragment was labeled by PCR amplification, with
the addition of [-32P]deoxycytosine triphosphate (800
Ci/mmol, 10 µCi/µL; Amersham, UK). Five microliters of the PCR
mixture was diluted with 25 µL of 1 g/L sodium dodecyl sulfate and 10
mmol/L EDTA. Five microliters of this dilution was mixed with 5 µL of
formamide dye (950 mL/L formamide, 20 mmol/L EDTA, 0.5 g/L bromphenol
blue, 0.5 g/L xylene cyanol FF). The PCR DNA was denatured by boiling
at 100 °C for 3 min, and then chilled immediately on ice. Samples (4
µL) were loaded onto a 7.5% polyacrylamide nondenaturing gel (ratio
of acrylamide to bisacrylamide 49:1) in 1x Tris–borate EDTA (TBE)
buffer, with 50 mL/L glycerol. Gels were 40 cm x 30 cm x 0.4 mm.
Electrophoresis was at 200 V for 16 h at room temperature and the
gel was then transferred onto Whatman 3-mm chromatographic paper,
dried, and exposed to hyperfilm ß max (Amersham, UK) for 12–24 h at
-70 °C before developing. The samples are run towards the anode
(+). The gel shows the pattern of fragments from 29 FH heterozygous
patients (14 in loading 1 and 15 in loading 2). From most samples, the
same two single-stand bands and a (more intense) faster migrating
double strand can be seen. The single and double strands of the first
and second loadings are designated respectively on right and left, SS1,
SS2, and DS, the double strands having migrated ~30 cm and the single
strands ~20 cm in the gel. SSCP variants are apparent in one sample
from the first loading and in one from the second loading, as indicated
by arrows. Since each sample is from a heterozygous
individual, these sample also show the normal bands, but fainter. For
these SSCPs, no heteroduplex bands were seen.
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Familial Hypercholesterolemia
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FH is characterized clinically by an increase in the concentration
of LDL cholesterol in blood, tendon xanthomata, and an increased risk
of myocardial infarction, and is present in 5–10% of individuals who
develop coronary artery disease under the age of 55 years in the UK and
the US (32)(33). On the basis of the estimated
population frequency of carriers of 1:500, there are >100 000 FH
heterozygous individuals in the UK, of which probably <3000 have been
identified to date. Once identified, the hyperlipidemia of these
patients is responsive to treatment by diet and drugs
(34), and such treatment reduces subsequent morbidity and
mortality (e.g., 35). Children who have inherited two
defective alleles of the LDL receptor occur at a frequency of one per
million of the population. In these children there is usually little
useful lowering of plasma LDL cholesterol concentrations in response to
diet or drugs, and many suffer a major coronary event in the first or
second decade of life, but life expectancy can be extended by
appropriate treatment (36).
FH results from different genetic defects in a cell surface receptor,
which normally controls the uptake of plasma LDL (1), or,
in a small proportion of patients, one particular defect in the
apolipoprotein (apo) B, the ligand for the receptor (37).
This disorder, which is called familial defective apo B, has been
reported to occur in ~3% of FH patients in the UK, and the mutation
in the apo B gene causing it can be easily detected by PCR and
allele-specific oligonucleotides.
For many heterozygous FH individuals, a clear diagnosis can be made on
the basis of increased plasma cholesterol. However, several studies
(e.g., 38) have shown that measures of total cholesterol or
LDL cholesterol do not allow unequivocal diagnosis of FH in 10–15% of
cases, and even measures of LDL receptor function on patients'
monocytes show overlap between the values obtained for some
"normal" individuals and patients with a defect in the receptor
(39). By contrast, a genetic approach to identify LDL
receptor defects gives an unequivocal result, and there is no doubt
that the equivocal nature of the tests currently available to identify
children with FH is one of the factors that deter some clinicians from
actively pursuing such diagnosis in the relatives of a patient with FH.
The advantage of an unequivocal DNA test would be both to allay fears
for half of the relatives, and to identify children for whom monitoring
dietary intake and appropriate therapy should be started.
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The LDL Receptor and LDL Receptor Gene Mutations
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The LDL receptor is a membrane protein of 839 amino acids that is
responsible for cholesterol uptake into cells via receptor-mediated
endocytosis of cholesterol-rich lipoproteins secreted by the liver
(32). The LDL receptor binds two different ligands, apo B,
which is the sole apoprotein of LDL, and apo E, which is found on the
triglyceride-rich lipoproteins and their remnants. Once the LDL
receptor has bound a ligand, it clusters in coated pits, where it is
taken up by the cell via endocytosis. The ligand is released from the
receptor in the lysosome and the receptor is then recycled to the cell
surface, where it can bind a ligand again. The human LDL receptor gene
was cloned and characterized >10 years ago (32). It is
located on the short arm of chromosome 19 (p13.1–p13.3) and, as shown
in Fig. 3
, spans 45 kb and comprises 18 exons and 17 introns. Five
classes of mutations at the LDL receptor locus have been identified on
the basis of the phenotypic behavior of the mutant protein, and to date
there have been >200 different mutations of the LDL receptor gene
characterized at the DNA level (reviewed in 40 and
41). They have given valuable insights into the function of
the different domains in the LDL receptor.
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Figure 3. Distribution of 207 point mutations and small
deletions/insertions in the LDL receptor gene in FH patients reported
worldwide (up to Jan. 1996) (41).
The intron–exon structure of the gene is indicated by boxes
(not to scale), and the function of the coding exons are indicated.
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Within a geographically or culturally isolated population, or where a
large proportion of people are related by descent because of migration,
there may be a single mutation causing FH in many of the patients (for
refs. see 40 and 41). In the UK, where there
is a very heterogeneous population, a priori it is unlikely that any
mutations will be present at a high frequency, and so far no mutation
detected has been present at a frequency >2–3% (41).
Because of the genetic diversity present, this is likely also to be the
case in most countries in Europe or in the US. Although it would be
feasible to develop methods to screen for reported mutations, our
calculations demonstrate that it is more cost and time effective to use
an approach such as SSCP that will allow any mutation in the
LDL receptor gene to be detected, rather than to screen specifically
for all mutations.
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Current Progress of SSCP Analysis on FH Research
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With the modifications in sample handling described above, it has
been possible to screen DNA from 800 FH patients for mutations in the
LDL receptor at the rate of one exon per week, which occupies 3.5
research assistant days/week. The LDL receptor gene has 18 exons, and
pairs of oligonucleotides and amplification conditions have been
reported (42) that allow the amplification of the promoter
plus coding portions of the entire gene in 22 fragments (exon 4 has to
be amplified in three parts and exon 10 in two parts because of their
size). In all cases the amplifying oligonucleotides are complementary
to intron sequences, which therefore also allow comparison of the
intron–exon boundary junction in patients, where mutations may cause
defects in correct splicing of nuclear RNA. This has resulted in >170
SSCPs identified at a rate of 10–12 per week.
In some situations, we wish to undertake a large number of different
PCRs simultaneously on the same DNA sample or on a small set, e.g.,
samples from four newly diagnosed FH patients. Instead of drying a
different template in each well, drying a different premixed pair of
PCR primers in each well is equally efficient in PCR yield
(42). This suggests that little of the oligonucleotide is
irreversibly bound to the plastic. The PCR premix is arranged to
contain a template DNA instead of a primer pair, and an example of an
SSCP autoradiograph from the simplified setup of 22 PCRs from the LDL
receptor gene in four patients with FH is shown in Fig. 4
. Typical PCRs in this laboratory are currently 20 µL, with 40
ng of template DNA and 8 pmol of each PCR primer. Eight picomoles of a
20-mer primer represents ~48 ng, so that a dried DNA template plate
contains 40 ng of DNA per well and a dried primer-pair plate contains
96 ng of oligonucleotide per well. Thus, on a weight basis, the layer
of DNA dried onto the plastic is of the same order for the two
strategies.
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Figure 4. Electrophoresis of PCR products from four
independent heterozygous FH patient DNAs, using dried arrays of PCR
primer pairs.
The PCR products are electrophoresed under SSCP conditions and are
grouped by PCR product, i.e., by exon number, except for exon 4 (3
fragments) and exon 10 (2 fragments). Almost all PCR reactions yielded
PCR product in all four samples. The lowermostband represents double-stranded DNA, the upper bands
single strands. There are SSCPs in exon 2 that probably are caused by a
common neutral (SfaN1) polymorphism and are not disease
causing, in exon 6 (probable mutation in sample 4), in exon 10 3'
(probable mutation in sample 3), and in exons 13 (known
AvaII polymorphism) and 15 (MspI polymorphism).
No mutation SSCP was detected in samples 1 and 2 in this run.
Confirmation of the cause of the SSCPs can be carried out by direct
sequencing of the PCR product (see text).
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Interpretation of DNA Results
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Once a sequence change in a gene has been identified by SSCP,
routine direct sequencing of amplified DNA is used to determine the
precise change and to confirm its likely deleterious effect on the
function of the protein. The ultimate proof that a mutation destroys
function can only come from in vitro expression studies, but in most
cases inspection of the mutation gives a clear indication of its likely
effect, for example a change creating a premature stop codon, a
frameshift mutation, or a large deletion or rearrangement of the gene
that results in the generation of a truncated protein is very likely to
be the cause of FH in this patient. In addition, a missense mutation
that alters a critical amino acid, for example one that changes or adds
a cysteine or some other nonconservative change, is very likely to
result in a defective LDL receptor and therefore to be the cause of FH
in this patient. However, more caution must be used with a missense
mutation that causes a conservative amino acid change or occurs in a
noncritical region of the protein. To date, all amino acid changes
detected in the LDL receptor except one have been associated with
functional effects, but since only FH patients have been studied
extensively, the sample is biased and the conclusion may not always be
true. The exception to this rule is the
alanine370
threonine change, with the threonine allele
occurring in 10% of healthy individuals and being associated with at
best a mild phenotype in men (43). Therefore, such
conservative missense mutations should be given a diagnosis of
probably causing FH in the patient, unless they have been
expressed in vitro and shown to cause either a defect in binding or in
the number of LDL receptor molecules on the surface of the cell.
Finally, with mutations in splice junctions, at intron–exon
boundaries, within the promoter or upstream region, within introns,
within the wobble position of amino acids, or within the 3'
untranslated region, formally, all such mutation must be treated as
possible or probable FH until expressed. However, where such a mutation
has been detected in more than one patient with FH, and where the
mutation occurs on different haplotypes (i.e., is an independent
mutation), and is not detected in the general population, such a
mutation can safely be designated as definite FH, but the predicted
effect on LDL receptor function must be made with care. Thus a mutation
in the conserved AG sequence that occurs within the intron side of the
exon–intron boundary is extremely likely to be causing a splice
defect, but it may not be possible to predict the exact effect on mRNA
splicing and therefore LDL receptor function.
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Future Developments in SSCP Mutation Screening for FH
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Some patients will have no defect identified by the SSCP
technique, although published data (e.g., 1) suggest that
the sensitivity of the method is extremely high (80–90%). Some
patients may have a major deletion or rearrangement of the gene, and in
the UK the frequency of gross alterations (insertions and deletions) is
~5% (44); this is likely to be similar in other
countries. Many of these gross alterations have occurred because of
recombination between repetitive Alu-type elements, and
detailed analysis indicates homologous recombination involving
Alu sequences as the mechanism of the rearrangements. In the
LDL receptor gene the deletions and insertions described are
distributed over the whole of the gene, so there are no isolated
hotspots that could be used to develop a simple rapid strategy for
gross rearrangement detection. When screening large numbers of patients
for mutations in the LDL receptor gene, Southern transfer will become
the last line of investigation after identification of point mutations
by faster and easier means, such as SSCP. In the case of already known
rearrangement, PCR-based tests can be constructed to identify patients
with that particular mutation. PCR-based tests may also eventually
replace Southern transfer for assays for general rearrangement,
analogous with methods established for the dystrophin (Duchenne) gene
(45).
A proportion of the patients with no detected SSCP may have been
misdiagnosed as having FH, but it is also possible that a (unknown)
proportion of patients may have hypercholesterolemia caused by a defect
in another gene. In such families it would be possible to use DNA
polymorphisms and a "cosegregation" approach to confirm or exclude
the inheritance of the LDL receptor gene with the hyperlipidemia
phenotype, and only depending on the results to identify relatives who
have inherited the defective allele of the LDL receptor gene. There are
several bi-allelic PCRable restriction fragment length polymorphisms
that can be used for this purpose (46), as well as some
hypervariable repeat polymorphisms (47). These
polymorphisms are also useful in haplotyping to prove recurrent
mutations.
At the present time the majority of detected mutations are novel,
indicating that general screening methods such as SSCP will continue to
be useful for FH for some time. However, in patients in the UK, ~30%
of all detected SSCPs occur in exon 4 and 18% in exon 3
(41). This suggests that a useful strategy will focus on
familial defective apo B and exons 3 and 4, and that this would lead to
the rapid detection of the specific mutation in 11–14% of patients.
In other parts of the world where founder mutations have been
identified, or if common mutations or "region-specific" mutations
are detected, it is possible that, as with mutations causing
thalassemia, a targeted or sequential approach to specific mutation
testing will be useful in the future.
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Acknowledgments
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I.N.M.D. was the recipient of a British Heart Foundation
Intermediate Fellowship and now holds a Lister Institute Fellowship.
S.E.H. is supported by a Chair award and Program grant RG16 from the
British Heart Foundation, and V.G. by the Icelandic Council of Science.
The work was also supported by the Sir Halley Stewart Trust, Helen
Eppel Fund, and John Pinto Foundation.
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Footnotes
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1 Nonstandard abbreviations: SSCP, single strand conformational polymorphism; FH, familial hypercholesterolemia; and apo, apolipoprotein. 
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Abstract
Introduction
