Rapid screening test of most frequent BRCA1/BRCA2 pathogenic variants in the NGS era

Abstract

The average risk of breast cancer in general Slovak population of women is 4-5% and the risk of ovarian cancer is 2%. Probability of breast/ovarian cancer development is higher in individuals carrying a causative germline DNA variant in BRCA1 or BRCA2 gene responsible for hereditary breast/ovarian cancer (HBOC). Although a major proportion of inherited breast/ovarian cancers are due to the mentioned causal mutations, a number of new genes have emerged. Here we describe a rapid, multiplex and comprehensive approach for the detection of pathogenic variants in BRCA1 and BRCA2 genes which most frequently occur in Slovak HBOC population. Analysis comprises the combination of mutation specific methods. Fluorescent PCR amplification followed by fragment analysis for detection of insertions/deletions in exon 11 of BRCA1 gene. Second method is SNaPshot analysis for detection of the most frequent missense and ins/del variants in exons 2, 5, 13, 20 of BRCA1 and exons 11, 23 and 25 of BRCA2 gene. Altogether, we have analyzed 687 samples, 86 (12.5%) in group 1, which fulfilled indication criteria based on the positive family/personal history. Group 2 involved 601 (87.5%) cases, who did not meet the indication criteria and only the screening test was recommended. Using the combined approach, we have identified 47 mutated samples (6.8%), 40 in group 1 (46.5%) and 7 in group 2 (1.1%). However, the presented screening test would not provide complex results of BRCA1/2 gene analysis, it offers testing accessible to a broader spectrum of individuals under the threshold of indication for whole gene analysis. This approach may provide valuable information even in the NGS analysis era.

The highest frequency of BRCA1 c.3700_3704del detected among Albanians from Kosovo

“…The c.3700_3704del mutation was reported in Greece with a frequency between 0.3%–1.5% in breast and ovarian cancer cases [ 12 , 25 ]. Additionally, the mutation was observed in breast and ovarian cancer patients from Turkey, Germany, Denmark, Romania and Slovakia with a frequency between 0.2–0.9% [ 26 – 30 ]. The observation of the BRCA1 c.3700_3704del mutation in many different populations raise the question of whether it is an ancient mutation with single origin, or whether it has arisen several times in human history.…”

The results of multigene panel sequencing in Slovak HBOC families

“…Since the establishment of genetic testing of breast/ovarian cancer susceptibility genes among HBOC families in Slovakia, several studies related to this issue have been reported so far [31][32][33][34][35][36][37][38][39].…”

“…)del) and 4 different PV in BRCA2 (c.9403del, c.7595_7596ins, c.4005dup, c.3076A>T). According to the previous studies, six most frequent variants in the BRCA1 gene (c.68_69del, c.181T>G, c.3700_3704del, c.4243del, c.5329dup, c.843_846del) and four most frequent variants in the BRCA2 gene (c.3076>T, c.5645C>A, c.9098dup, c.9403del)account for the majority of BRCA1/2 mutation spectrum in Slovak HBOC families[38,39]. ,800 years ago in the region of Northern Europe, and spread to the various European populations, including the Ashkenazi Jewish population during the following centuries[48].…”