Short communication
Asymmetric real-time PCR detection of BRCA1 5382insC mutation by melting curve analysis in the LightCycler

https://doi.org/10.1016/j.cca.2007.12.024Get rights and content

Abstract

Background

5382insC BRCA1 frameshift mutation is a common founder mutation for many populations worldwide and a high-risk allele for the development of hereditary breast and/or ovarian cancer. Our goal was to develop a novel, reliable and rapid method for its detection.

Methods

We developed an asymmetric real-time PCR method with hybridization probes in the LightCycler. Genotyping was performed by melting curve analysis.

Results and conclusions

The developed method was in concordance with reference methods when tested in 85 peripheral blood and 107 tumor DNA samples from Greek breast and/or ovarian cancer patients. The described method proved to be simple, cost-effective, easy to perform and rapid enough for routine use as a screening method in high-risk families and especially in the Greek, Slavic and Jewish populations where 5382insC mutation is the most common BRCA1 mutation.

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)

There are more references available in the full text version of this article.

Cited by (17)

View all citing articles on Scopus
View full text