Clinical Chemistry 43: 24-29, 1997;
(Clinical Chemistry. 1997;43:24-29.)
© 1997 American Association for Clinical Chemistry, Inc.
Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis
Elizabeth M. Rohlfs1,
William G. Learning1,
Kenneth J. Friedman2,
Fergus J. Couch3,
Barbara L. Weber3,4 and
Lawrence M. Silverman1,a
1
Department of Pathology and Laboratory Medicine, CB7600, and
2
Curriculum in Genetics, University of North Carolina, Chapel Hill, NC 27599.
3
Department of Medicine, Room 1009, 422 Curie Blvd.,
and
4
Department of Genetics, University of Pennsylvania,
Philadelphia, PA 19104.
a Author for correspondence. Fax 919-966-4526; e-mail lmsmol.dhl1{at}mail.unch.unc.edu
 |
Abstract
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The tumor suppressor genes BRCA1 and BRCA2,
which confer increased susceptibility to breast and (or) ovarian
cancer, were recently identified. Mutation analysis of
BRCA1 has demonstrated significant allelic heterogeneity;
however, some distinct mutations have been detected in unrelated
individuals. The most notable is the 185delAG mutation, which occurs at
an estimated frequency of ~1% in individuals of Ashkenazi Jewish
descent [1]. Although consensus has not been reached
regarding clinical testing for mutations in BRCA1, a tiered
strategy may be appropriate, in which direct testing for the more
common mutations is one component. Specific alleles can be detected by
using PCR-mediated site-directed mutagenesis (PSM), which alters the
PCR products derived from either the wild-type or mutant allele to
create or destroy a restriction endonuclease recognition site.
Recognition sites are introduced by a base substitution in one of the
primers. The alleles are then resolved by electrophoresis of the
digested PCR products. We have applied this technique to the detection
of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and
R1443X. Another mutation, 1294del40, can be resolved from the wild-type
allele by high-resolution gel electrophoresis alone. The PSM technique
is sensitive, does not require radioactivity, and is specific for
individual mutations.
Key Words: indexing terms: polymerase chain reaction • ovarian cancer • genetics
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Introduction
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Breast cancer afflicts >180 000 women in the US every year, and
5–10% of these cases are estimated to result from an inherited
mutation in one of at least two genes (2). The
BRCA1 gene was linked to chromosome 17q21 in 1990
(3) and was isolated in October 1994 (4).
About half of the early-onset breast cancer families, and 80% of the
early-onset breast and ovarian cancer families, are thought to reflect
mutations in BRCA1 (5). Women with germline
mutations in BRCA1 have an estimated lifetime risk for
breast cancer of 85% and a 20–50% risk for ovarian cancer
(6). Moreover, individuals who carry a BRCA1
mutation apparently have a fourfold increased risk for colon cancer,
and carrier men have a threefold increased risk for prostate cancer
(6).
BRCA1 is thought to be a tumor suppressor gene that encodes
a protein capable of negatively regulating tumor growth. The gene spans
>100 000 bp and has 22 coding exons. One exon (exon 11) accounts for
>60% of the coding sequence. Mutations are distributed throughout the
coding sequence with no apparent clustering or hot spots
(7). About 70% of the distinct mutations identified to
date are small deletions or insertions that cause a frameshift in the
coding sequence of the gene and result in a premature stop codon.
Including nonsense mutations, ~86% of the distinct mutations
detected so far are predicted to result in a truncated or shortened
protein because of a premature termination of translation
(8). The function of the protein product is unknown, but
it contains a RING finger motif thought to be involved in
protein–protein interaction or nucleic acid binding (i.e., a
transcription factor). Recently, the protein product has also been
shown to contain a granin motif and may be secreted from the cell
(9).
Analysis of BRCA1 in individuals with a strong family
history suggestive of inherited disease has identified recurrent
mutations in unrelated individuals as well as mutations that appear to
be limited to particular families. More than 100 different mutations
have been identified to date (8). The 185delAG mutation
has been detected in multiple individuals, most of whom are of
Ashkenazi Jewish descent
(1)(8)(10). Among unrelated
members of this ethnic group, the 185delAG mutation has been found on
the same haplotype, indicating a likely founder effect
(10)(11)(12)(13). However, other BRCA1
mutations, such as R1443X and 4184del4, have been found on different
haplotypes—suggesting either de novo occurrence of these mutations in
distinct populations or significant recombination near or within the
BRCA1 gene (11)(12).
Given the large number of distinct mutations identified, many of which
have been found only once, development of a protocol for mutation
detection should include both mutation scanning methods and methods
that detect specific mutations. Recurrent BRCA1 mutations
have been detected by direct methods such as allele-specific
oligonucleotide hybridization (1)(14) and
allele-specific polymerase chain reaction (PCR) (15).
PCR-mediated site-directed mutagenesis (PSM) (16)
identifies specific alleles by altering a sequence to introduce or
remove a restriction site. The sequence is modified by substituting (or
mismatching) a base near the mutation of interest in one of the two PCR
primers. Thus, amplification of the region containing the mutation site
will result in incorporation of the base change in the PCR product.
After digestion with the appropriate restriction enzyme and
electrophoretic separation of the fragments, the alleles can be
identified. This technique is amenable to the detection of small
insertions, deletions, nonsense and missense mutations and has been
utilized for analysis of several genes, including those for cystic
fibrosis transmembrane conductance regulator (CFTR)
(17), low-density lipoprotein receptor (18),
and phenylalanine hydroxylase (19).
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Materials and Methods
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patients' samples
Samples were obtained from families at high risk for breast and
(or) ovarian cancer, with three or more individuals with early-onset
breast cancer or two with breast cancer and one with ovarian cancer.
The mutations had been previously characterized by single-strand
conformation polymorphism analysis or by conformation-sensitive gel
electrophoresis and had been confirmed by sequencing. The K562 human
cell line (chronic myelogenous leukemia, from ATCC, Rockville, MD) was
used as a negative control.
DNA was extracted either directly from peripheral blood lymphocytes or
from Epstein–Barr virus-transformed lymphoblastoid cell lines by using
standard methods or the Puregene DNA isolation kit (Gentra Systems,
Minneapolis, MN) according to the manufacturer's directions.
pcr
Primers were designed to either introduce or destroy a restriction
site in either the wild-type or the mutant allele. To do this, we
mismatched, or substituted, a base in the sequence of a primer that
anneals adjacent to the mutation locus; this way, we could generate
products with the corresponding base change (Fig. 1
). The primer sequences used to detect each mutation are shown
in Table 1
. The 50-µL PCR reactions contained 10 mmol/L Tris-HCl (pH
8.4), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 1.5 U of
Taq polymerase (Perkin-Elmer, Norwalk, CT), 1 µmol/L each
of the forward and reverse primers, 200 µmol/L deoxynucleotides, and
100 ng of template DNA. The amplification conditions were 95 °C for
2 min followed by 35 cycles of: 30 s at 95 °C, 30 s at
62.5 °C, and 1.0 min at 72 °C. The reactions were ended with a
10-min incubation at 72 °C. We used a Perkin-Elmer 9600 thermal
cycler for all amplification reactions.
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Figure 1. Primer design for mutation detection.
The mismatched bases are indicated by asterisks, the mutated
bases by open boxes, and the restriction endonuclease sites
by shaded boxes.
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restriction endonuclease digestion
After the amplification, the PCR products were digested with the
appropriate restriction endonucleases (New England Biolabs, Beverly,
MA). The digestion reactions contained: 25.5 µL of PCR product, 3.0
µL of buffer (supplied with the enzyme), and 1.5 µL of enzyme (10
U). These components were incubated overnight at 37 °C. After 6 µL
of 5x loading buffer (i.e., 300 mL/L glycerol and 10 g/L bromphenol
blue in 10 mmol/L Tris, pH 8.0, plus 1 mmol/L disodium EDTA) was added
to the digestion reaction, 15 µL of the digest was loaded on a 10%
nondenaturing polyacrylamide gel and electrophoresed for 2 h at
325 V. The gel was stained with ethidium bromide reagent (0.5 mg/L in
134 mmol/L Tris, pH 8.6, containing 80 mmol/L boric acid and 30 mmol/L
disodium EDTA) and transilluminated with ultraviolet light to visualize
the bands.
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Results
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Amplification and digestion of PCR products spanning the 185delAG
mutation are shown in Fig. 2
. Both the 185delAG allele and the wild-type allele are normally
cut by DdeI because the deleted AG is either preceded or
followed by another AG, thus preserving the restriction site. However,
when the C at nucleotide 188 of the noncoding strand is changed to a T
by the mismatched reverse primer, the cutting site is destroyed in the
mutant allele. Therefore, PCR amplification, followed by restriction
enzyme digestion, produces fragments of 150 and 26 bp from the
wild-type allele and an uncut fragment of 176 bp from the mutant allele
(Table 2
). The 26-bp fragment migrates quickly and is not seen on the
gel. The pairing of single-strand PCR products, one of which contains
the 2-bp deletion, results in the formation of two heteroduplex
molecules (20). The heteroduplex molecules have
electrophoretic mobilities different from those of the homoduplex
molecules and migrate above the 176-bp fragment, at ~181 and 189 bp.
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Figure 2. PSM detection of mutations in BRCA1.
After normal and mutant alleles were amplified by PCR, the products
were digested with the appropriate restriction endonuclease and
separated by electrophoresis as described in the text. For all gels:
lane M, HaeIII-digested  X174 DNA size markers
in basepairs; lane 1, individual heterozygous for the
indicated mutation; lane 2, negative control; and lane
3, water blank.
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Using the same mismatch primer strategy for 5382insC, introducing an A
into the sequence at nucleotide 5384 destroys a BstNI site
in the wild-type allele. The restriction site is still present in the
mutant allele even with the mismatched base because the mutant C is
inserted into a string of 3 Cs. Digestion of the mutant product results
in 250- and 23-bp fragments (Fig. 2
). Visible in the heterozygote
sample are both the digested fragment of 250 bp from the mutant allele
and the full-size product of 273 bp from the wild-type allele.
The nonsense mutation E1250X results from a G
T transversion at
nucleotide 3867 and changes a Glu to a stop codon
(8)(21). This base change creates a natural
cutting site for RmaI. However, because this enzyme has not
been cloned and is therefore relatively costly, we introduced a
StyI site into the mutant PCR product. The StyI
site is introduced by using the reverse primer to mismatch the A at
nucleotide 3870 of the anti-sense strand with a C. The full-length PCR
product is 321 bp and its presence indicates the wild-type allele,
whereas amplification and digestion of the mutant allele yields
fragments of 300 and 21 bp (Fig. 2
). Again, the heterozygote sample
shows both fragments.
The R1443X mutation results from a CT base transition at nucleotide
4446 and changes an Arg to a stop codon (21). Introduction
of an AlfIII site into the PCR product from the mutant
allele results in fragments 180 and 17 bp long after restriction
digestion (Fig. 1
). The wild-type allele product is not digested but
stays at 197 bp (Fig. 2
). Another base substitution (CG) at
nucleotide 4446 has also been reported (8)(21)
but is a benign polymorphism. This base change will not be detected by
digestion with AlfIII, and the analysis will still indicate
the presence of a wild-type allele. Alternatively, the sensitivity of
PSM at this locus can be increased by designing the assay to produce a
BstU1 cutting site in the wild-type allele. With this
approach, virtually any base change at that particular locus will
prevent the creation of the restriction site, and the analysis will
indicate a non-wild-type allele. Although this design is more sensitive
(i.e., can detect more base changes), it is not specific for a
particular mutation.
A 40-bp deletion beginning at nucleotide 1294 can be detected by PCR
amplification across the deletion and subsequent high-resolution gel
electrophoresis. Our primer set amplifies a PCR product of 300 bp from
the wild-type allele and a product of 260 bp from the mutant allele.
Amplification of DNA from an individual heterozygous for this mutation
produces the two expected bands as well as a heteroduplex band that
migrates significantly more slowly (at ~2300 bp) than either
homoduplex molecule (Fig. 2
).
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Discussion
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Except for a few disorders such as sickle cell anemia and
1-antitrypsin deficiency, most single-gene
disorders are characterized by significant allelic heterogeneity. More
than 500 different mutations that cause cystic fibrosis have been
identified in the CFTR gene, and >170 different mutations
that cause phenylketonuria have been identified in the phenylalanine
hydroxylase gene (22)(23). In addition, many
of these mutations are limited to individual families. At present, we
do not yet know how many different mutations will be identified in
BRCA1, and a comprehensive protocol for mutation detection
is thus difficult to formulate. Methods such as single-strand
conformation polymorphism, denaturing gradient-gel electrophoresis, or
the protein truncation test have proven useful for scanning coding
regions and critical intron sequences for sequence variants in a
variety of disorders (24)(25)(26). After screening
large numbers of individuals, investigators have found that, among
unrelated individuals with some disorders, certain mutant alleles are
found more frequently than others. These recurrent alleles may be
detected by using direct PCR-based methods such as allele-specific
oligonucleotide hybridization (1)(14),
allele-specific PCR (15), or PSM (17).
We have applied PSM to the detection of four mutations found in
BRCA1. This technique is suitable for detecting single-base
changes, small deletions, and small insertions. Because detection is
based on alteration of restriction endonuclease recognition sites, PSM
is highly specific for the mutation of interest when the assay is
designed to cut the mutant allele. For this reason, the PCR products
from the mutant alleles E1250X, 5382insC, and R1443X contain the
restriction sites. When a constitutive cutting site is present within
the PCR product, the analytical sensitivity of the assay is increased
because the constitutive site acts as a control for enzyme activity.
Designing a PSM for 185delAG was somewhat more difficult because of the
repetitive AG and thus required digestion of the wild-type allele.
Therefore, any alteration in the sequence that disrupted the
DdeI recognition site would prevent digestion and indicate a
non-wild-type allele. In this respect, the assay became more sensitive
but less specific.
PSM analysis can also be affected when a mutation other than the one in
question happens to alter the region where a primer should anneal. For
example, there have been several reports of an 11-bp deletion in
BRCA1 beginning at nucleotide 188
(4)(8)(13). This mutation will
prevent the 185delAG reverse primer from annealing and thus will
prevent amplification from the mutant allele; the results will indicate
the presence of only a wild-type allele. Also, a 4-bp deletion within
the E1250X reverse primer binding site may prevent amplification or
indicate a heteroduplex formation if amplification does occur. To the
extent that confounding mutations are subsequently found to be
moderately prevalent, PSM assays may be readily designed for these
mutations.
The combination of PSM and high-resolution gel electrophoresis allows
us to detect five BRCA1 mutations, two of which (185delAG
and 5382insC) are the mutations detected most frequently (Table 3
) (8). R1443X and 1294del40 are also recurrent
mutations but at a lesser frequency, and E1250X has been identified
only twice (8)(21). As an allele-specific
detection technique, PSM offers many advantages over other methods. It
does not require the use of radioisotopes, it is relatively cost
effective, and it does not require a subjective evaluation—as
sometimes occurs with oligonucleotide hybridization methods. In
addition, PSM is extremely flexible. With >150 restriction enzymes
commercially available, almost all single-base changes and small
deletions and insertions can be unequivocally identified.
Use of a direct test, such as PSM, to detect the more common mutations
may be a useful component of a tiered testing program (Fig. 3
). Initial screening of a patient's sample for the five
mutations described here would detect ~28% of the characterized
BRCA1 alleles (8). If no mutation was detected
by PSM, a gene-scanning technique could be used to screen for less
common, private mutations. The scanning technique chosen would depend
on the types of mutations that occur in the gene (i.e., protein
truncation test to detect truncating mutations, heteroduplex analysis
to detect missense mutations). Any putative mutations identified by the
scanning techniques would be confirmed by sequencing.
The technical aspects of mutation detection have now advanced
sufficiently to permit analysis of any portion of the human genome. We
strongly recommend judicious use of detection strategies, such as those
described here, especially when analyzing human DNA for mutations that
may predispose an individual to an adult-onset disorder. Although it is
not within the scope of this paper to discuss all the relevant social
(i.e., insurance and employment discrimination) and scientific (i.e.,
penetrance of particular alleles, polymorphism vs mutation) issues
involved with clinical genetic testing, we recommend to the reader
several recent articles that discuss these topics
(27)(28).
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Acknowledgments
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This work was supported in part by National Institutes of Health
grant NRSA CA62588 to F.J.C.
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Abstract
Introduction
