Short communicationAsymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler
Introduction
The tumor suppressor gene BRCA1 is located on chromosome 17 and is linked to hereditary breast and ovarian cancer. BRCA1 protein has a significant role in the signalling of DNA damage and in DNA repair. Mutations are widely distributed throughout the whole gene and confer to mutation carriers a lifetime risk of 82% for breast cancer and 54% for ovarian cancer [1], [2].
Our group is involved in BRCA1 mutation screening in Greece [3], [4]. We have found that the single-base insertion 5382insC in exon 20 of BRCA1 resulting in frameshift and a truncated protein, is the most common mutation of this gene among Greek breast and ovarian cancer families since it was found in 7 out of the 85 families that we have studied [3]. In the same group of patients, another four scattered BRCA1 and another three scattered BRCA2 mutations were also detected; therefore 5382insC represents the majority of BRCA1 mutations in Greece. This mutation is also present in high frequencies in Eastern–South Eastern Europe and Ashkenazi Jews [5], [6], [7]. Up to now, 5382insC is the second most common mutation in the BRCA1 gene according to the Breast Information Core (BIC) database http://research.nhgri.nih.gov/projects/bic (1063 hits, Dec07). So far only heterozygotes have been detected for this mutation; the mutation is located in a critical area of the corresponding protein resulting to the inadequacy of mutant homozygotes to survive. Here, we describe a novel, rapid and reliable method that is based on real-time PCR and melting curve analysis that can easily distinguish between wild-type and mutant 5382insC heterozygotes samples.
Section snippets
Patients
After obtaining informed consent, peripheral blood from 85 index cases of breast and/or ovarian cancer families were obtained from various hospitals in Greece. These samples were analyzed by PTT (Protein Truncation Test) and DNA sequencing for the whole BRCA1 gene as previously described [3]. Seven of these patients were found to be positive heterozygotes for the BRCA1 5382insC mutation (8.2%, 7/85). This sample set was used for the initial development of the real-time PCR method in the
Results
Real-time PCR amplification for the BRCA1 5382insC detection proved quite robust. The PCR product was free from other by-products or primer-dimers and at its correct size (232 bp) as judged by agarose gel electrophoresis. By using asymmetric PCR (forward/reverse primer ratio: 1/5), we have increased the template copies for the probes to anneal and therefore the emitted fluorescence signal. Therefore, the sensitivity of the method was increased by allowing even a 60 pg DNA sample to be
Discussion
Intense optimization of both asymmetric real-time PCR reactions for the detection of BRCA1 5382insC frameshift mutation led to very good amplification efficiencies and a significant Tm difference between the peaks of normal and mutant alleles so that easy identification of mutants of even low amount of DNA could be performed. To increase the cost-effectiveness of the detection, we propose the use of the wt-rxn for screening purposes in order to identify any sequence alterations underneath the
References (14)
- et al.
Germ line BRCA1 & BRCA2 mutations in Greek breast/ovarian cancer families: 5382insC is the most frequent mutation observed
Cancer Lett
(2002) - et al.
Presence of high-risk human papillomavirus sequences in breast cancer tissues and association with histopathological characteristics
Clin Biochem
(2006) - et al.
A rapid and sensitive approach to mutation detection using real-time polymerase chain reaction and melting curve analyses, using BRCA1 as an example
Mol Diagn
(1999) - et al.
Detection of a single base substitution in a single cell using the LightCycler
J Biochem Biophys Methods
(2001) - et al.
Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2
Science
(2003) - et al.
Breast and ovarian cancer
N Engl J Med
(2003) - et al.
Genetic counseling of medullary breast cancer patients
Clin Genet
(2004)
Cited by (17)
Mutation scanning of exon 20 of the BRCA1 gene by high-resolution melting curve analysis
2010, Clinical BiochemistryPrognostic significance of RASSF1A promoter methylation in operable breast cancer
2009, Clinical BiochemistryContribution of BRCA1 5382insC mutation to triplene-gative and luminal types of breast cancer in Ukraine
2022, Breast Cancer Research and TreatmentSOX17 promoter methylation in plasma circulating tumor DNA of patients with non-small cell lung cancer
2016, Clinical Chemistry and Laboratory MedicineCox-2 and brca1 have altered expression profile in different canine tumors
2016, Journal of Animal and Plant Sciences