Clinical Chemistry 45: 976-981, 1999;
(Clinical Chemistry. 1999;45:976-981.)
© 1999 American Association for Clinical Chemistry, Inc.
BRCA1 Gene Mutations in Sporadic Ovarian Carcinomas: Detection by PCR and Reverse Allele-specific Oligonucleotide Hybridization
Dan Tong1,
Margit Stimpfl1,
Alexander Reinthaller1,
Norbert Vavra1,
Silvia Müllauer-Ertl2,
Sepp Leodolter1,3 and
Robert Zeillinger1,a
1
Department of Obstetrics and Gynecology, Division of Gynecology, and
2
Department of Clinical Pathology, General Hospital of Vienna, University of Vienna, A-1090 Vienna, Austria.
3
Ludwig-Boltzmann Institute for Oncology and Fertility
Treatment, A-1090 Vienna, Austria.
a Address correspondence to this author at: General Hospital of Vienna, Department of Obstetrics and Gynecology, Molecular Oncology Group, Währinger Gürtel 18-20, EBO 05, A-1090 Vienna, Austria. Fax 43-1-40400-7832; e-mail robert.zeillinger{at}akh-wien.ac.at
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Abstract
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Background: Although germline mutations in BRCA1 play
a central role in familial breast and ovarian cancers, to date, no
somatic mutations in BRCA1 have been reported in sporadic
breast cancer, and only five somatic mutations have been identified in
the sporadic ovarian carcinomas. Because loss of heterozygosity appears
frequently at the BRCA1 locus in nonfamilial
breast and ovarian carcinomas, we searched for mutations in the
BRCA1 gene in sporadic ovarian tumors.
Methods: We developed a detection system based on PCR and
reverse allele-specific oligonucleotide hybridization on membrane
strips for the simultaneous detection of 17 frequently occurring
mutations in the BRCA1 gene.
Results: As little as 2% mutant DNA in a sample could be
detected. Two of 122 DNA samples isolated from sporadic ovarian tumor
biopsies contained the Cys61Gly mutation. Both mutations were germline
mutations. One of these was an ovarian metastasis of a primary
fallopian tube carcinoma. The tubal carcinoma was also confirmed to
contain the Cys61Gly mutation.
Conclusions: This is the first report that a germline
BRCA1 mutation is associated with primary tubal carcinoma.
The 17 specific mutations in the BRCA1 gene do not play
a major role in the tumorigenesis and progression of sporadic ovarian
cancer.© 1999 American Association for Clinical Chemistry
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Introduction
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The BRCA1 gene contains 22 coding exons and encodes a
nuclear phosphoprotein of 1863 amino acid residues
(1)(2). In human breast cancer cell lines and
spermatocytes, BRCA1 protein binds to Rad51, which functions in DNA
recombination and repair, suggesting a role for BRCA1 specifically
during mitosis and meiosis (3). Recent studies have
suggested that BRCA1 contributes to cell-cycle arrest and growth
suppression through the induction of p21 (4). BRCA1 and p53
cooperatively induce apoptosis of cancer cells, and BRCA1 may
coordinately regulate gene expression together with p53 as tumor
suppressors (5).
The BRCA1 gene frequently is mutated in familial breast
and/or ovarian cancers. To date, >500 distinct mutations,
polymorphisms, and unclassified variants have been identified [Breast
Cancer Information Core database (BIC), 1998]. Most small deletions,
insertions, and point mutations lead to premature termination of
translation and, thus, truncated proteins. Germline mutations in
BRCA1 are thought to be responsible for ~45% of familial
breast cancers and for >80% of the inherited breast and ovarian
cancer syndrome (6). Although BRCA1 mutations are
involved in the etiology of hereditary breast and/or ovarian cancers,
they seem to play no important role in the tumorigenesis of
sporadic breast and ovarian cancer. To date, no somatic mutations in
BRCA1 have been reported in sporadic breast cancer, and only
five somatic mutations have been identified in sporadic ovarian
carcinomas (7)(8). However, this is not
consistent with the observations that loss of heterozygosity frequently
appears at the BRCA1 locus in nonfamilial breast and ovarian
carcinomas (9)(10). Therefore, we were
interested in searching for mutations in the BRCA1 gene in
sporadic ovarian tumors.
At present, mutation screening in BRCA1 has been performed
mostly by the use of the single-strand conformation polymorphism
method, the protein truncation test, allele-specific oligonucleotide
(ASO) hybridization, denaturing gradient gel electrophoresis,
and direct sequencing (11)(12)(13)(14). These techniques have
different detection limits. Because tumor biopsies usually contain
varying proportions of healthy tissue, it is questionable
whether the very low rate of BRCA1 mutations found in
ovarian carcinomas is attributable to the detection limits of these
techniques. Thus, we chose reverse ASO hybridization, a method that has
been reported to detect as little as 2.5% mutant DNA (15).
Reverse ASO was originally developed to facilitate the simultaneous
screening of different allelic variants at an amplified locus
(16). Because BRCA1 is a large gene, screening
for all possible mutations through the whole coding sequence is
time-consuming and costly. The results of mutation screening in the
BRCA1 gene suggest that different mutations have different
carrier frequencies. Certain mutations have been found repeatedly,
whereas others have been reported only once. We chose 17 frequently
occurring mutations in BRCA1, which covered >50% of all
reported mutations when this study was initiated (BIC, 1996). We
developed a system for the simultaneous detection of these mutations by
the use of PCR and reverse ASO hybridization techniques and evaluated
the system, using DNA samples containing known mutations. The detection
limit of the system was tested with control plasmids containing cloned
BRCA1 fragments. We then screened 122 DNAs from sporadic
ovarian carcinomas for the existence of these mutations.
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Materials and Methods
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tumor samples
Fresh biopsies from 122 sporadic ovarian carcinomas (without known
family histories) and the corresponding EDTA blood samples were
collected in the Department of Obstetrics and Gynecology, University of
Vienna, from 1994 to 1997. The histologic diagnoses (17) of
the 122 patients are shown in Table 1
.
dna preparation
We isolated DNA from blood or tumor tissue, using
commercially available kits (DNA Extraction System I and II;
ViennaLab). Three DNA samples with different known
mutations were obtained from the Department of Pathology, University of
Cambridge, Cambridge, UK.
pcr
A total of 17 mutations in the BRCA1 gene were chosen
because of their prevalence (Table 2
, based on data from BIC, 1996). The sense and antisense
primers for the amplification of the fragments containing these
mutations are shown in Table 2
. All antisense primers were labeled with
biotin. DNA (50 ng) from peripheral blood or tumor biopsy was used as
the template for PCR in a total volume of 50 µL. The reaction mixture
included 25 pmol each of the sense and antisense primers, 250 µmol/L
dNTPs (ViennaLab), 5 µL of 10x amplification buffer (10 mmol/L
Tris-HCl, pH 9.0, 50 mmol/L KCl, 0.1 g/L gelatin, 1.5 mmol/L
MgCl2, and 1.0 mL/L Triton X-100;
ViennaLab) and 1.6 U of Super Taq Polymerase (HT Biotechnology). PCR
was performed in a Perkin-Elmer GeneAmp PCR system 9600 with 40 cycles
at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for
30 s. All reactions were preceded by a primary denaturation step
at 94 °C for 1 min. The PCR product (10 µL) was then resolved on
4% agarose gels containing 0.1 mL/L SYBR Green I (Molecular
Probes). The gels were visualized with 254 nm transillumination and
photographed.
construction of control plasmids and evaluation of the detection
limit
To evaluate the system, plasmids containing cloned mutant
fragments for each tested mutation were constructed using the
overlapping extension PCR technique (Fig. 1
). Briefly, two separate PCRs were carried out to generate two
overlapping fragments. One of these fragments contained the mutation at
the one end, whereas the other fragment contained the mutation at the
other end. The bands of these two PCR products were excised from the
agarose gel, placed in H2O, and vortex-mixed.
Portions of the solutions were combined, dNTPs and Taq polymerase were
added, and 10 cycles of extension were carried out as follows: 30
s at 94 °, 30 s at 50 °, and 30 s at 72 °C. After
the extension was completed, the sense and antisense primers were
added, and a PCR of 30 cycles of 30 s at 94 °C, 30 s at
50 °C, and 30 s at 72 °C was performed. The mutant PCR
product and the PCR product generated from wild-type DNA were cloned
into plasmids (TA cloning kit; Invitrogen). To evaluate the
detection limit, plasmid DNA containing the wild-type fragment was
mixed at different ratios with plasmid DNA containing the corresponding
mutant fragment. PCR was carried out as described above.
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Figure 1. Construction of mutation controls.
Step 1, two separate PCRs were performed to generate two
overlapping fragments that contained the specific mutation in the
overlapping region; step 2, the two PCR products were
excised from the agarose gel and combined; step 3, dNTPs
and Taq polymerase were added, and extension was performed for 10
cycles. After the extension was completed, the sense and antisense
primers were added to the reaction, and a PCR of 30 cycles was carried
out.
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reverse allele-specific hybridization
Oligonucleotides were designed to specifically hybridize with
either wild-type or mutant PCR products (Table 3
). The oligonucleotides were 5' labeled with an
NH2 group and immobilized on nylon membranes
(15). Oligonucleotides were applied as lanes, and the
membranes were cut into strips, producing a barcode-like pattern of the
oligonucleotides. The biotinylated PCR products containing wild-type
sequences and possible specific mutations were hybridized to the
membrane strips, using the VARISTRIP Detection Assay kit (ViennaLab).
This kit provides alkaline denaturation buffer, a hybridization
mixture, wash buffers, a streptavidin-horseradish peroxidase complex,
and color developer containing p-nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate.
sequencing
To confirm the mutations detected by reverse ASO hybridization, we
sequenced PCR products, using a 310 Genetic Analyzer and
BigDyeTM Terminator Cycle Sequencing Kit (Applied
Biosystems).
histopathology
The specimens were obtained from adnexectomy. Tissues were fixed
in buffered formalin and embedded in paraffin. The sections were
routinely stained with hematoxylin and eosin.
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Results and Discussion
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Examples of the detection of the PCR products of BRCA1
by reverse ASO hybridization are shown in Figs. 2
and
3. As indicated, PCR products from both mutant and wild-type
plasmids hybridized specifically with the corresponding
oligonucleotides on the membrane strips. PCR products from DNA
containing known mutations also specifically hybridized to the
corresponding oligonucleotides. The aim of this study was to detect
several possible mutations simultaneously. Therefore, it was important
to optimize PCR and ASO hybridization conditions to allow the
amplification and detection of fragments containing different mutations
in a single assay. PCR optimization can be achieved by selecting
primers with similar melting temperatures and amplicons with similar
lengths and base composition. The specific hybridization of the PCR
products to the immobilized oligonucleotides was dependent on the
length of the hybridization oligonucleotides, the positions of
mutations in the oligonucleotides, the temperature for hybridization
and washing, and the salt concentration in the washing buffer. Because
several different hybridization oligonucleotides were immobilized on a
strip, the stringency of the hybridization could be adjusted only by
the length and base composition of the oligonucleotides. We tested
several oligonucleotides that differed in length and in the
positions of the mutations within the oligonucleotides to obtain clear
positive signals and to avoid cross-hybridization with nonspecific PCR
products. The hybridization process took <2 h, and the results could
be visualized directly from the strips. In our system, the
oligonucleotides specific for both wild-type and mutant alleles of all
17 selected mutations were divided into three groups and immobilized on
three different strips. We could screen 96 samples for all 17 mutations
within 1 week. Therefore, this system is very useful to screen for
known mutations in many samples. It is also appropriate for the
detection of BRCA1 mutations in familial breast and ovarian
carcinomas as a prescreening method before analysis of the whole coding
sequence.
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Figure 2. Examples of hybridization results with PCR products
generated from plasmid DNAs containing the wild-type or the mutant
fragment for four different mutations.
Strip 1, 185delAG and 188del11 wild-type
plasmid; strip 2, 185delAG mutant plasmid;
strip 3, 188del11 mutant plasmid; strip
4, 1294del40 wild-type plasmid; strip 5,
1294del40 mutant plasmid; strip 6, 5382insC wild-type
plasmid; strip 7, 5382insC mutant plasmid; strip
8, H2O control. The oligonucleotides immobilized at
the corresponding positions on the strips are indicated on the
left (wt, wild-type; mut,
mutant).
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To evaluate the detection limit of this system, we chose three
mutations that represented a short deletion (3450del4), an insertion
(1135insA), and a single-base exchange (E908X). As shown in Fig. 4
, this system can detect as little as 2% mutant DNA in DNA
samples. Thus, this combined PCR-reverse ASO hybridization method for
the simultaneous detection of several known mutations is also
applicable for mutation analysis in tumor samples containing healthy
tissue. In addition, this method may be used for mutation
detection in genes other than BRCA1.
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Figure 4. Evaluation of the detection limit.
Strips 1–9 represent mixed DNAs containing
90%, 50%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0% mutant plasmid
DNA, respectively; strip 10, H2O control.
The oligonucleotides located at the corresponding positions on the
strips are indicated on the left (wt,
wild-type; mut, mutant).
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Two of the 122 sporadic ovarian carcinomas evaluated contained the
Cys61Gly mutation in exon 5 of BRCA1. However, analysis of
the corresponding DNA extracted from peripheral blood confirmed that
these two mutations were germline mutations. Further histopathological
analysis revealed that one of these ovarian carcinomas was a metastasis
of a primary fallopian tube carcinoma. The dilated tube was filled with
papillary and solid tumor mass penetrating the muscularis as well as
spreading to the ovary. The neoplasm was composed of fine branching
papillae covered by one or more layers of epithelium with enlarged
pleomorphic and hyperchromatic nuclei with increased and abnormal
mitoses. In some areas, the tumor exhibited solid sheets of cells with
small or large foci of necrosis. The tumor biopsy from the tubal
carcinoma was also confirmed to contain the Cys61Gly mutation by both
reverse ASO hybridization and direct sequencing. To our knowledge, this
is the first report that a BRCA1 mutation is
associated with primary tubal carcinoma. To date, there have been no
systematical studies on the BRCA1 mutations and fallopian
tube carcinomas. However, a recent study suggested that serous tubal
carcinoma may have a molecular pathogenesis similar to those of serous
ovarian and uterine carcinomas because these three types of carcinomas
have strikingly similarities in the frequency and pattern of
chromosomal changes (18).
At the initiation of this study in 1996, the 17 selected mutations
accounted for >50% of all described mutations. However, many novel
mutations have been found since then. Thus, the 17 analyzed mutations
represent only 30% of the mutations published to date (BIC, February
1999). Although we analyzed 122 sporadic ovarian carcinomas, we could
not detect any mutations other than the two germline mutations
described above. Thus, the 17 specific mutations in the
BRCA1 gene do not play a major role in the tumorigenesis and
progression of sporadic ovarian cancer.
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Figure 3. Examples of mutation detection in tumor samples.
Strips 1 and 2, wild-type DNA
probes; strips 3, 4, and
5, DNA samples from known carriers of the 185delAG,
1294del40, and 5382insC mutations, respectively. The oligonucleotides
located at the corresponding positions on the strips are indicated on
the left (wt, wild-type;
mut, mutant).
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Acknowledgments
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This project was supported by the Anniversary Fund of the Austrian
National Bank for the Promotion of Scientific Research and Teaching
(ÖNB 5572 and ÖNB 6056). We thank Drs. Simon Gayther and
Bruce Ponder of the Department of Pathology, Cambridge
University, for providing us with three control DNA samples with
known BRCA1 mutations. We also thank Eva Schuster for
excellent technical assistance.
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Top
Abstract
Introduction
