Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5 breakpoint in intron 4; 3 breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G>C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies

Spectrum and characterisation of BRCA1 and BRCA2 deleterious mutations in high-risk Czech patients with breast and/or ovarian cancer

<p>Abstract</p> <p>Background</p> <p>The incidence of breast cancer has doubled over the past 20 years in the Czech Republic. Hereditary factors may be a cause of young onset, bilateral breast or ovarian cancer, and familial accumulation of the disease. <it>BRCA1 </it>and <it>BRCA2 </it>mutations account for an important fraction of hereditary breast and ovarian cancer cases. One thousand and ten unrelated high-risk probands with breast and/or ovarian cancer were analysed for the presence of a <it>BRCA1 </it>or <it>BRCA2 </it>gene mutation at the Masaryk Memorial Cancer Institute (Czech Republic) during 1999–2006.</p> <p>Methods</p> <p>The complete coding sequences and splice sites of both genes were screened, and the presence of large intragenic rearrangements in <it>BRCA1 </it>was verified. Putative splice-site variants were analysed at the cDNA level for their potential to alter mRNA splicing.</p> <p>Results</p> <p>In 294 unrelated families (29.1% of the 1,010 probands) pathogenic mutations were identified, with 44 different <it>BRCA1 </it>mutations and 41 different <it>BRCA2 </it>mutations being detected in 204 and 90 unrelated families, respectively. In total, three <it>BRCA1 </it>founder mutations (c.5266dupC; c.3700_3704del5; p.Cys61Gly) and two <it>BRCA2 </it>founder mutations (c.7913_7917del5; c.8537_8538del2) represent 52% of all detected mutations in Czech high-risk probands. Nine putative splice-site variants were evaluated at the cDNA level. Three splice-site variants in <it>BRCA1 </it>(c.302-3C>G; c.4185G>A and c.4675+1G>A) and six splice-site variants in <it>BRCA2 </it>(c.475G>A; c.476-2>G; c.7007G>A; c.8755-1G>A; c.9117+2T>A and c.9118-2A>G) were demonstrated to result in aberrant transcripts and are considered as deleterious mutations.</p> <p>Conclusion</p> <p>This study represents an evaluation of deleterious genetic variants in the <it>BRCA1 </it>and <it>2 </it>genes in the Czech population. The classification of several splice-site variants as true pathogenic mutations may prove useful for genetic counselling of families with high risk of breast and ovarian cancer.</p

Prevalence of BRCA1/2 mutations in sporadic breast/ovarian cancer patients and identification of a novel de novo BRCA1 mutation in a patient diagnosed with late onset breast and ovarian cancer: implications for genetic testing

In order to adequately evaluate the clinical relevance of genetic testing in sporadic breast and ovarian cancer patients, we offered comprehensive BRCA1/2 mutation analysis in patients without a family history for the disease. We evaluated the complete coding and splice site regions of BRCA1/2 in 193 sporadic patients. In addition, a de novo mutation was further investigated with ultra deep sequencing and microsatellite marker analysis. In 17 patients (8.8%), a deleterious germline BRCA1/2 mutation was identified. The highest mutation detection ratio (3/7 = 42.9%) was obtained in sporadic patients diagnosed with breast and ovarian cancer after the age of 40. In 21 bilateral breast cancer patients, two mutations were identified (9.5%). Furthermore, 140 sporadic patients with unilateral breast cancer were investigated. Mutations were only identified in patients diagnosed with breast cancer before the age of 40 (12/128 = 9.4% vs. 0/12 with Dx > 40). No mutations were detected in 17 sporadic male breast cancer and 6 ovarian cancer patients. BRCA1 c.3494_3495delTT was identified in a patient diagnosed with breast and ovarian cancer at the age of 52 and 53, respectively, and was proven to have occurred de novo at the paternal allele. Our study shows that the mutation detection probability in specific patient subsets can be significant, therefore mutation analysis should be considered in sporadic patients. As a consequence, a family history for the disease and an early age of onset should not be used as the only criteria for mutation analysis of BRCA1/2. The relatively high mutation detection ratio suggests that the prevalence of BRCA1/2 may be underestimated, especially in sporadic patients who developed breast and ovarian cancer. In addition, although rare, the possibility of a de novo occurrence in a sporadic patient should be considered