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"Thanks to new Multiplicom test kits physicians are able to identify all the genetic mutations of a condition at once, and use this information to initiate the right - personalized - treatment.”

Poster abstract EuroMedLab Athens 2017: High resolution melting analysis is very useful to identify BRCA1 c.4964_4982del19 (rs80359876) founder calabrian pathogenic variant on peripheral blood and buccal swab DNA

Different BRCA1 founder mutations are confined to geographically isolated regions or specific populations. BRCA1 5083del19 mutation is recurrent and specific to individuals of Italian descent with a founder effect on the Calabrian population. The correct identification of this type of mutation is one of pitfalls of NGS mainly due to insufficient coverage, read lenght or alignment quality. In view of this, we set up a rapid, low cost, high-throughput High Resolution Melting Analysis (HRMA) for genotyping the Italian BRCA1 5083del19founder mutation starting from peripheral blood and/or buccal swab DNA. 



DNA samples were obtained from 30 subjects, 15 wild type (WT) and 15 mutated (M) for the BRCA1 5083del19, previously amplified by BRCA MASTR Dx (Multiplicom,Niel, Belgium), defined by NGS on the Illumina MiSeq® platform (Illumina Inc., San Diego, CA, USA) and confirmed by Sanger sequencing. To validate the assay, we performed HRMA analysis on a set of further 20 unknown samples, obtained from Italian HBOC patients, belonging to Calabria region. We also evaluated the sensitivity by mixing the heterozygote sample for BRCA1 c.4964_4982del19 variant and a reference WT DNA sample at ratios of 50 %, 10 %, 2.5 % of the mutated sample. PCR-HRMA were performed on the LightCycler® 480 Real-Time PCR System. Data, analyzed with LightCycler 480 GeneScanning Software version 1.2 (Roche Diagnostics), were normalized, temperature-shifted and converted to a derivative plot for analysis. Melting temperatures (Tms) were derived at the greatest dF/dT value of the derivative curve data. 


WT (95bp) and M amplicons (76bp) showed a clearly different melting profile; furthermore the WT and M samples presented evident differences in Tm (TmWT=79.3± 0.5; TmM =74.3±0.5). So, HRMA results were 100 % concordant with direct sequencing. In addition, HRMA sensitivity appear superior to direct sequencing, allowing the detection of heterozygous sequence changes up to 2.5 % of the mutated allele: this result is particularly promising above all when this assay could be applied to DNA from FFPE tumor samples. 


We provide evidence about application of HRMA in unambiguously genotyping of the founder BRCA1 c.4964_4982del19 variant in individuals belonging to Calabria Italian region. In fact, HRMA was confirmed to be particularly suitable for the identification of BRCA1 c.4964_4982del19 variant, making this approach useful in clinical molecular diagnostics.
Authors: M. De Bonis, A. Minucci, E.D. Capoluongo, 
Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy (Italy) 
Click here for the original poster abstract.
Click here for more information on the BRCA MASTR DX.