Inmaculada de Juan Jiménez

Advantages of the high resolution melting in the detection of BRCA1 or BRCA2 mutation carriers.

OBJECTIVE The aim of the study is to explore the reliability of the high resolution melting (HRM) analysis for the identification of BRCA1/BRCA2 mutation carriers among the family members of index patient (IP) and for distinguishing the presence of two or more genetic variants within the same amplicon. DESIGN AND METHODS We studied 27 different BRCA1/BRCA2 pathogenic mutations detected in 35 families with 194 subjects. HRM was performed in the LightCycler 480 (Roche). RESULTS HRM method detected 110 BRCA1/BRCA2 mutations among the 192 relatives studied (57%). No false negative results were observed in any of the family members and all of them were in agreement with sequencing analysis, therefore the method might help to avoid unnecessary sequencing of wild type (WT) genotypes. The HRM method also allows the detection of other alterations that we initially had not searched (three unclassified variants and several polymorphisms). Furthermore, HRM has also been capable of distinguishing the presence of two or more genetic variants in the same amplicon of the same sample. CONCLUSIONS HRM is a rapid, sensitive, specific, cost-effective and reliable screening method that in less than 2 h allows the easy identification of BRCA1 and BRCA2 genetic variations and also avoids the unnecessary sequencing of WT genotypes. Furthermore the method is also capable of detecting new genetic variants and allows the simultaneous detection of the presence of more than one genetic variant.

Advantage of high-resolution melting curve analysis over conformation-sensitive gel electrophoresis for mutational screening of BRCA1 and BRCA2 genes.

BACKGROUND Mutation screening of BRCA1 and BRCA2 (BRCAs) genes is a time-consuming and costly procedure that demands faster and cheaper alternative methods for routine diagnostics. The present study is aimed at comparing the results obtained with screening mutations methods, conformation sensitive gel electrophoresis (CSGE) and high-resolution melting analysis (HRMA), for BRCAs attending to their specificity, sensitivity, reliability and cost-efficiency. METHODS We included 52 DNA samples of index patients from high-risk families. The mutational screening was performed by CSGE according to the Ganguly (1993) method and HRMA according to a modified De Leeneer (2008) method. The assays were performed in 384 well plates in the LightCycler 480 (Roche). All PCR products showing altered patterns were confirmed by sequencing. RESULTS The results obtained with the mutational study of BRCAs genes showed that HRMA exhibited higher sensitivity than CSGE as it was able to detect a wide mutational spectra of genetic variants in a larger number of samples. Aditionally, the combination of HRMA with hybiridization probes in a second step of the assay allows the specific confirmation of mutations. Furthermore, HRMA use less time, allowing the reduction of analysis time. CONCLUSIONS HRMA offers clear advantages over CSGE for the mutation screening of BRCAs genes as it has greater sensitivity and higher efficiency and it is less time-consuming.

Broad BRCA1 and BRCA2 mutational spectrum and high incidence of recurrent and novel mutations in the eastern Spain population.

It is well known that the pathogenic mutations in BRCA1 or BRCA2 genes are detected in 5–10% of total breast (BC) and ovarian cancer (OC) and in 20–30% of BC/OC found in families with strong history of cancer [1]. However, the incidence of the BRCA1 and BRCA2 pathogenic mutations depends on the criteria adopted to select the families to be studied [2], and the mutation spectrum varies considerable due to the influence of the ethnic groups [3]. In addition, ethnicity could substantiate the appearance of founder mutations coming from an old ancestor such as the recurrent mutations detected among Ashkenazim [4, 5]. The Program of Genetic Counselling in Cancer of Valencia Community was launched in March 2005 and we reported the results of the first 147 nonrelated families included, describing the preliminary mutational spectrum [6] and novel mutations [7]. However, since then, the number of subjects studied for BRCA1 and BRCA2 mutations has increased considerably having reached the 704 families at the end of 2008. This large number of individuals prompted us to update our series and to establish a consistent mutational spectrum of the population of Eastern Spain identifying the recurrent mutations and relevance of the novel mutations. We studied 704 index patients (IP) (689 females and 15 males) who enrolled in the Program of Genetic Counselling in Cancer of the Valencian Community from March 2005 to December 2008. The IPs were selected by the Units of Genetic Counselling in Cancer (UGCC) according to the inclusion criteria established in the Program of Genetic Counselling in Cancer for familial BC/OC [8, 9]. All the individuals signed a written consent elaborated by the Conselleria de Sanitat (Valencia Community) in accordance with the Helsinki Declaration (1964, amended in 1975 and 1983) [10]. BRCA1 and BRCA2 mutations were detected by amplifying all the exons and the exon–intron boundaries of both genes by PCR using the primer pairs and the PCR conditions reported in the Breast Cancer Information Core (BIC) [11]. To identify the genetic variations, we carried out a pre-screening of the heteroduplexes of the PCR products by conformation sensitive gel electrophoresis (CSGE) [12] followed by the sequencing of the PCR products in which heteroduplexes were identified. In BRCA1, we found 26 different deleterious mutations in 62 IPs (17 frameshift, 3 splicing, 4 nonsense, and 2 missense mutations; Table 1); 16 of these mutations have been This study was conducted on behalf of the Group for Assessment for Hereditary Cancer of Valencian Community.