Advantage of high-resolution melting curve analysis over conformation-sensitive gel electrophoresis for mutational screening of BRCA1 and BRCA2 genes
Introduction
About 5–10% of breast or ovarian cancers (BC/OC) are caused by germline mutations in the breast cancer genes BRCA1 (OMIM 113705) or BRCA2 (OMIM 600185) [1]. After the BRCA1 and BRCA2 genes (BRCAs) were identified by Miki et al. (1994) [2] and Wooster et al. (1995) [3] genetic testing became available and is now routinely offered to women from high-risk families. Accordingly, since the implantation of the Program of Genetic Counselling in Cancer in the Valencian Community (Spain) in 2005 [4] the genetic studies of BRCAs mutations were performed in individuals with a strong familial history of BC or OC.
A wide variety of methods have been used for the screening of these genes [5]. Direct sequencing allows the characterization of the alteration sequence and is considered the gold standard method. However, the direct sequencing of BRCAs, due to its large size and absence of hot-spots, is a time-consuming and costly procedure [6]. Therefore, there is a need for faster and less expensive alternative methods for BRCAs routine diagnostics with comparable accuracy. The most widely used methods are the denaturing high-pressure liquid chromatography (DHPLC), conformation-sensitive gel electrophoresis (CSGE) and fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE) [5].
In our laboratory, the main reference laboratory for BRCAs mutation studies in the Valencian Community (Spain), mutational pre-screening was carried out by the heteroduplex screening of the PCR products by CSGE [7]. However, this method requires post-PCR treatment including gel electrophoresis, being time-consuming and an expensive procedure.
High-resolution melting analysis (HRMA) is a new and attractive gene scan tool that quickly performs PCR and identifies sequence alterations without requiring post-PCR treatment [8]. For BRCAs mutations there are several reports that support the utility of HRMA as a fast high-thoughoutput method for the scanning of these genes [9], [10], [11], [12]. The most comprehensive report is that written by De Leeneer et al. (2008) [12], which scanns the entire coding regions and flanking boundaries of both genes.
Since there is a lack of reports comparing HRMA with other screening methods, we have aimed in the present study to compare the results of our conventional CSGE screening method for the detection of mutations in BRCAs genes with the new one of HRMA in the terms of sensitivity, specificity, reliability and cost-efficiency.
Section snippets
Patients and samples
We include 52 DNA samples from index patients (in 49 screening BRCA1 and BRCA2 and in three only screening BRCA2) with a strong family history of BC/OC recruited by the Units of Genetic Counseling of Valencia Community. All the subjects were informed of the significance of genetic testing and signed a written consent elaborated by the Consellería de Sanitat (Valencia Community Health Services) in accordance with the Helsinki Declaration [13].
We performed a blind assay, in which all samples had
Results and discussion
The mutational screening of BRCA1 has been achieved in 49 DNA samples and 35 different genetic variants were found between the two methods; 27 of them were amplified by both methods (Table 1). HRMA detected any genetic variant in all the 49 samples, whereas CSGE only in 39 of them. CSGE detected 27 different genetic variants in 305 PCR products, whereas HRMA detected 27 different genetic variants in 340 PCR products (Table 1).
Both methods are consistent in the detection of three of the five
Acknowledgements
This work has been funded by the “Instituto de Salud Carlos III” under Grant PI06/0505. We would like to thank the help and support given by the “Oficina del Plan del Cáncer. Dirección General de Salud Publica. Consellería de Sanitat. Comunidad Valenciana.” We also express our gratitude to the “Instituto de Investigación Sanitaria- Fundación Hospital La Fe” for having granted Inmaculada de Juan (Bch Sc and Specialist in Clinical Chemistry) in a research project on sporadic breast cancer and the
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On behalf of the Group for Assessment of Hereditary Cancer of Valencia Community.