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Technical Briefs |
1 Laboratoire d’Oncologie Moléculaire Humaine, Centre Oscar Lambret, 3 rue Frédéric Combemale, BP 307, 59020 Lille Cédex, France
aaddress for correspondence: fax 33-3-2029-5535, e-mail jp-peyrat{at}o-lambret.fr
Approximately 5% of breast cancers are considered as hereditary, and their development is associated with germline mutations of specific genes. The BRCA1 gene was isolated in 1994 (1) and is estimated to account for almost one-half of inherited breast cancers and three-fourths of inherited breast/ovarian cancers. BRCA1 is a large gene containing 5592 nucleotides spread over 
100 000 bases of genomic DNA.
More than 1200 different mutations have been found in BRCA1 associated with breast or ovarian cancer (2)(3). Mutations, recurring or unique in distribution, are dispersed throughout the coding sequence. Recurring mutations are either international or national. Six mutations have been observed commonly (3): 185delAG, 5382insC, 300T>G, 4446C>T, 4184delTCAA, and 3875delGTCT. Other frequent mutations have been localized to specific countries: 5149delCTAA (4) and 3958delCTCAGinsAGGC (5) in France; 2804delAA in Holland (6)(7); 3960 C>T in northern France, Belgium, and Holland (8); and 3600del11 in northeastern France (9).
As no functional assays are available, detection of BRCA1 mutations must be carried out at the DNA level. Because the gene is large and the mutations are distributed throughout its length, systematic sequencing is certainly the "gold standard" method. However, this method is time-consuming and expensive.
We describe here a method (10), multiplex single-nucleotide primer extension (MSNPE) analysis, to simultaneously detect 11 international and regional recurring mutations.
The patients were treated at the Centre Oscar Lambret for breast and/or ovarian cancer and belonged to high-risk families. Written informed consent was obtained from each patient. Genomic DNA was extracted from 2 mL of citrate blood samples by use of the QIAamp DNA Blood Midi Kit (Qiagen).
Fragments of BRCA1 gene containing the 11 recurrent screened mutations were amplified by multiplex PCR. The PCR mixture (total volume, 25 µL) contained 15 mM Tris-HCl (pH 8.0), 50 mM KCl, 2 mM MgCl2, 0.4 mM each of the deoxynucleotide triphosphates (Amersham Biosciences), 100 ng of genomic DNA, 2 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems), and the primers (see Table 1
in the online Data Supplement that accompanies the online version of this Technical Brief athttp://www.clinchem.org/content/vol50/issue1/) at 1.6 µM for exon 17, 0.8 µM for exons 2 and 13, 0.4 µM for exons 5 and 11H, 0.2 µM for exon 11F, and 0.12 µM for exon 20. After activation of AmpliTaq Gold DNA polymerase for 10 min at 95 °C, the thermocycling conditions were 55 cycles at 94 °C for 30 s, 54 °C for 20 s, and 72 °C for 2 min, with a final elongation step at 72 °C for 8 min.
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PCR products were purified by use of Sephacryl 400 (Amersham Biosciences) in Costar microplates and digested by incubation for 1 h at 37 °C with exonuclease I (Amersham Biosciences) and shrimp alkaline phosphatase (Roche Diagnostics).
The MSNPE reaction was performed with the ABI Prism SnaPshot Multiplex reagent set (Applied Biosystems). The 11 purified primers (Genosys; Table 1
), each specific for a screened mutation, bind to the complementary sequence in the presence of fluorescently labeled dideoxynucleotide triphosphates (ddNTPs), and the AmpliTaq DNA Polymerase extends the primer by adding a single ddNTP to its 3' end. Fluorescent dyes are assigned to the individual ddNTPs as follows: green for A, yellow for C (which for convenience is black on the electropherogram), blue for G, and red for T. The reaction mixture contained 3 µL of PCR products, 5 µL of multiplex reaction premixture (fluorescently labeled ddNTPs, AmpliTaq DNA polymerase, and reaction buffer), and 2 µL of primers (0.5 pmol for mutation 3875delGTCT; 5 pmol for 185delAG, 300T>C, 2804delAA, 3600del11, 3960C>T, 4446C>T, and 5382insC; and 10 pmol for the others). The reaction was performed as follows: 27 cycles at 96 °C for 10 s, 50 °C for 5 s, and 60 °C for 30 s.
Samples were treated with 1 U of shrimp alkaline phosphatase at 37 °C for 1 h, then diluted 10-fold with deionized formamide and denatured at 95 °C for 5 min. The fluorescently labeled fragments were resolved by capillary electrophoresis on an automated ABI Prism 310 Genetic Analyzer (Applied Biosystems).
The specificity of the multiplex PCR was checked by electrophoresis of the products on 4% agarose gels containing ethidium bromide (see Fig. 1
in the online Data Supplement).
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The MSNPE reaction was performed on samples previously analyzed by direct sequencing. All mutations were easily detected. Fig. 1
shows the electropherograms of the wild-type DNA and the three most frequent BRCA1 mutations (3). Because all of the BRCA1 samples with mutated DNA were in a heterozygous state, two peaks were detected.
It is noteworthy that these two peaks did not overlap, although the sizes of their products were similar. This is a consequence of the electrophoretic mobility of these small final extension products, which depends not only on their lengths, but also on their nucleotide sequences and on the fluorescent dye used in the reaction.
To validate the procedure, we analyzed 48 DNA samples (24 samples with a BRCA1 mutation and 24 without). All of the mutations were always detected, and no false-positive results were observed in samples without mutations, demonstrating the reliability of the procedure. Repetitive analyses demonstrated the reproducibility of the procedure.
The present procedure was accurate: All 48 DNA samples already genotyped by direct DNA sequencing displayed concordant results. The 11 heterozygous mutations were easily detected when compared with the wild-type profile. The criteria of specificity for a "mutant" peak were as follows: (a) a peak was observed with the expected size and color; (b) the obtained peak was at least threefold higher than the background peaks of the same color; and (c) the wild-type peak was decreased.
For each screened mutation, the primer was designed to anneal immediately adjacent to the nucleotide at the mutation site, on either the sense or antisense DNA strand. The primer orientation allowed multiplex PCR in the absence of intra- and intercomplementarity. Despite the purification of the primers, several small peaks appeared, which seem to be extension products coming from n-1 oligonucleotides for the higher molecular weight primers corresponding to the 3958, 3875, and 4446 loci. These peaks were easily identified on all electropherograms.
The present procedure allowed the detection of 36% of the cases identified in our region and 32% of the international mutations (3). It could also be adapted easily and quickly by other laboratories to take into account their local mutation frequencies. In this regard, this procedure could potentially be a useful tool for prescreening studies.
The MSNPE reaction developed here requires only one reaction per patient sample, and it is possible to analyze 96 DNA samples in 1.5 working days. The subsequent interpretation of peak patterns is simple. This method is sensitive, rapid, and offers reduced laboratory costs. The multiplex technique increases both the practicability and ease of handling in the laboratory. If multichannel capillary electrophoresis instruments are used, this procedure could also be automated for high-throughput genotyping. Finally, a similar strategy can easily be developed to detect the most frequent BRCA2 mutations.
Acknowledgments
This work was supported by the Ligue Nationale Contre le Cancer (Paris, France), its local Committee (Lille, France), and the A.R.C. (Villejuif, France). We are grateful to Dr. Dave Fernig (University of Liverpool, Liverpool, UK) for critical reading of the manuscript.
References
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