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Technical Briefs |
1
Departments of Laboratory Medicine and Pathobiology;
2
Genetic Repository, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4;
3
Department of Pathology and Laboratory Medicine and Centre for Cancer Genetics, Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5;
4
Medicine and Pediatrics (Genetics), University of Toronto, Toronto, Ontario, Canada M5G 1L5;
a address correspondence to this author at: Department of Laboratory Medicine and Pathobiology, Rm. 402, Banting Institute, 100 College St., University of Toronto, Toronto, Ontario, Canada M5G 1L5: fax 416-978-5650, e-mail davidec.cole{at}utoronto.ca
BRCA1 and BRCA2 are tumor suppressor genes that are inactivated during neoplastic development (1)(2). Germline mutations of the two genes are transmitted in the autosomal dominant fashion and predispose carriers to the development of ovarian and/or breast cancers (3)(4). Mutations in BRCA1 are present in approximately one-half of the early-onset breast cancer families and 80% of the early-onset breast and ovarian cancer families (5), whereas BRCA2 mutations are believed to account for a comparable percentage of inherited breast cancer cases (6). Women with germline mutations in BRCA1 have a lifetime risk of 85% and up to 50% for breast and ovarian cancers, respectively.
In individuals of Ashkenazi Jewish background, mutations such as 185delAG and 5382insC in BRCA1, and 6174delT in BRCA2 are present in higher frequencies than other mutations because of founder effects (6)(7)(8). In one study, the three mutations accounted for 62% of Ashkenazi patients with ovarian and/or breast cancer (7). The high frequency of these mutations in the BRCA genes indicates their potential role in identifying individuals at risk.
Many methods have been reported for the study of BRCA mutations, including allele-specific oligonucleotide hybridization (8)(9), allele-specific PCR (10), PCR-mediated site-directed mutagenesis (11)(12), heteroduplex analysis (HDA) (13)(14)(15), single-strand conformation polymorphism (14)(16), and the protein truncation test (14)(15). Methods such as HDA, single-strand conformation polymorphism, and the protein truncation test, which are suitable for screening uncharacterized mutations, are relatively more laborious, whereas allele-specific PCR, PCR-mediated site-directed mutagenesis, and allele-specific oligonucleotide hybridization are more efficient for screening of specific characterized mutations.
Here we describe a simple and rapid method for the simultaneous
detection of three common mutations: 185delAG and 5382insC in
BRCA1, and 6174delT in BRCA2. Blood samples were
obtained with written informed consent. DNA was extracted from
peripheral blood cells as described previously (17), coded,
and analyzed in a blind fashion. Allele-specific oligonucleotide
primers were designed on the basis of published BRCA1 and
BRCA2 sequences (18)(19) and checked
against the NIH database (http://www.ncbi.nlm.nih.gov/Blast/) for
possible false priming. For each mutation, three primers (one common,
one specific for the mutant, and one specific for the wild-type allele)
were used. The competing mutant and wild-type primers were designed to
differ by ~20 bp in size, allowing easy detection of the PCR products
by routine electrophoresis and ultraviolet illumination after
ethidium bromide staining. The mutant (long) and wild-type (short)
primers both contain a mismatched base sequence near the 3' end. In the
early cycles of amplification, the mismatched sequences generate
mutagenized PCR products that are refractory to cross-amplification by
the competing primer, thereby ensuring specificity of the reaction
(20). The long (mutant) primer also incorporates two
additional mismatched bases at two contiguous positions corresponding
to the 5' end of the short (wild-type) primer. During the final cycles
of the PCR reaction, heteroduplexes may be formed from the short and
long products, but the contiguous mutagenized sequences in the long
product prevent filling up of the short product by using the long
strand as template. As a result, the mutant and wild-type products are
separated mutagenically. The primer sequences and sizes of
corresponding amplicons are shown in Table 1
.
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PCR amplification was carried out using the GeneAmp® PCR 2400 system from Perkin-Elmer. In each PCR reaction, 25 ng of genomic DNA was added to 10 µL of reaction mixture consisting of 1x PCR reaction buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 0.01 g/L gelatin), 3.25 mmol/L MgCl2, 0.2 mmol/L dNTPs, and 50 kU/L AmpliTaq GoldTM (all from Perkin-Elmer). Allele-specific primers were added at 2.0 µmol/L for P1 and P3; 0.4 µmol/L for P2; 0.12 µmol/L for P4, P5, and P6; 0.31 µmol/L for P7 and P9; and 0.24 µmol/L for P8. Each PCR reaction consisted of an initial 12 min of AmpliTaq Gold activation at 95 °C, followed by 35 cycles of 15 s of denaturation at 94 °C, 15 s of annealing at 57 °C, and 30 s of extension (with an increment of 1 s for each subsequent cycle) at 72 °C, and a final extension step of 5 min at 72 °C. At the conclusion of the reaction, the PCR product was mixed with 2 µL of loading dye (200 g/L sucrose, 0.05 g/L bromphenol blue) and then separated on precast Clearose BG Mini (9 x 12 cm) Gels (Elchrom Scientific) by routine submerged electrophoresis (120 V, 80 min). The resolved amplicons were then stained with 0.5 mg/L ethidium bromide and viewed under ultraviolet illumination.
Like other PCR techniques, mutagenically separated PCR (MS-PCR)
requires careful optimization of each reaction condition, including
magnesium concentration, cosolvents (dimethyl sulfoxide or
glycerol), and length and temperature of cycling stages. More
importantly, the concentrations (relative and absolute) of individual
primers must be determined empirically to give relatively equal
amplification of the wild-type and mutant alleles. If a mutation is
present in one of the alleles, two bands will be present. For the
185delAG mutation, the mutant and wild-type amplicons are 354 and 335
bp, whereas those of 5382insC are 295 and 271 bp, and those of 6174delT
are 171 and 151 bp. Therefore, a minimum of three bands (absence of any
mutant allele) and a maximum of six bands (all three mutations) can be
detected. Fig. 1
shows the electrophoretogram with patterns for zero to three
mutations present in a single sample. Because at least one of the two
allelic PCR products is present, MS-PCR provides an intrinsic quality
control against false negatives or PCR refractory conditions. The
presence of wild-type and mutant allelic products also allows easy and
objective interpretation of test results.
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We evaluated this assay using a cohort of 66 Ashkenazi Jewish women at an increased risk for breast/ovarian cancer (all have a blood relative diagnosed with epithelial ovarian cancer). The genetic status of this cohort of patients had previously been ascertained by HDA (15) and DNA sequencing. Using the optimized procedure, we identified 7 individuals with the 185delAG mutation, 4 with the 5382insC mutation, 3 with the 6174delT mutation, and 52 without any of the three mutations. The results were identical to HDA and DNA sequencing, indicating the reliability of MS-PCR technique. Overall, the multiplex approach allows easy and simultaneous detection of the three mutations. This assay eliminates the need for radioisotopes, endonuclease digestion, and high-resolution electrophoresis. We conclude that this method is simple, reliable, and can be considered for routine use.
Acknowledgments
We thank the Ontario Cancer Genetics Network and those who participated in the study.
Footnotes
* Pak Cheung Chan is a recipient of a postdoctoral fellowship in Clinical Chemistry and Laboratory Medicine from the Ontario Ministry of Health, Canada 
References
The following articles in journals at HighWire Press have cited this article:
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  J. Rogozinska-Szczepka, B. Utracka-Hutka, E. Grzybowska, B. Maka, E. Nowicka, A. Smok-Ragankiewicz, H. Zientek, J. Steffen, and A. Wojciechowska-Lacka BRCA1 and BRCA2 mutations as prognostic factors in bilateral breast cancer patients Ann. Onc., September 1, 2004; 15(9): 1373 - 1376. [Abstract] [Full Text] [PDF]
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