Assessing the RNA effect of 26 DNA variants in the BRCA1 and BRCA2 genes

Menéndez, Mireia; Castellsague, Joan; Mirete, Marc; Pros, Eva; Feliubadaló, Lídia; Osorio, Ana; Calaf, M; Tornero, Eva; Valle, Jesús Del; Fernández-Rodríguez, Juana; Quiles, Francisco; Salinas, Mónica; Velasco, Ángela; Teulé, Àlex; Brunet, Joan; Blanco, Ignacio; Capellà, Gabriel; Lázaro, Conxi

doi:10.1007/s10549-011-1661-5

Cited by 21 publications

(16 citation statements)

References 23 publications

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“…Normal mRNA splicing was observed for the remaining 5 variants of group B, including BRCA1 c.5333A>G and BRCA2 c.9116C>T, already analyzed [20]–[22] and BRCA1 c.548−3delT, c.594−4A>G, c.4097G>A, not previously characterized. To account for the possible occurrence of NMD, LCLs carrying these variants were analyzed following treatment with puromycin.…”

Section: Resultsmentioning

confidence: 66%

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Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations

et al. 2013

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Several unclassified variants (UVs) have been identified in splicing regions of disease-associated genes and their characterization as pathogenic mutations or benign polymorphisms is crucial for the understanding of their role in disease development. In this study, 24 UVs located at BRCA1 and BRCA2 splice sites were characterized by transcripts analysis. These results were used to evaluate the ability of nine bioinformatics programs in predicting genetic variants causing aberrant splicing (spliceogenic variants) and the nature of aberrant transcripts. Eleven variants in BRCA1 and 8 in BRCA2, including 8 not previously characterized at transcript level, were ascertained to affect mRNA splicing. Of these, 16 led to the synthesis of aberrant transcripts containing premature termination codons (PTCs), 2 to the up-regulation of naturally occurring alternative transcripts containing PTCs, and one to an in-frame deletion within the region coding for the DNA binding domain of BRCA2, causing the loss of the ability to bind the partner protein DSS1 and ssDNA. For each computational program, we evaluated the rate of non-informative analyses, i.e. those that did not recognize the natural splice sites in the wild-type sequence, and the rate of false positive predictions, i.e., variants incorrectly classified as spliceogenic, as a measure of their specificity, under conditions setting sensitivity of predictions to 100%. The programs that performed better were Human Splicing Finder and Automated Splice Site Analyses, both exhibiting 100% informativeness and specificity. For 10 mutations the activation of cryptic splice sites was observed, but we were unable to derive simple criteria to select, among the different cryptic sites predicted by the bioinformatics analyses, those actually used. Consistent with previous reports, our study provides evidences that in silico tools can be used for selecting splice site variants for in vitro analyses. However, the latter remain mandatory for the characterization of the nature of aberrant transcripts.

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Section: Resultsmentioning

confidence: 66%

“…Numerous studies have shown that these tools may be used to predict whether BRCA1 and BRCA2 mutations located at splice sites and adjacent regions are expected to have an effect on mRNA splicing [13]–[18], [20]–[22], [24]–[27]. Therefore, they have been proposed to be instrumental in UV classification.…”

Section: Introductionmentioning

confidence: 99%

Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations

et al. 2013

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“…There are 4 instances of inconsistent or conflicting splicing results (6, 8, 14, 16-19). These include BRCA1 c.212+3A>G, c.670+8C>T, and c.736T>G and BRCA2 c.517-19C>T (4, 19-25).…”

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confidence: 99%

“…These include BRCA1 c.212+3A>G, c.670+8C>T, and c.736T>G and BRCA2 c.517-19C>T (4, 19-25). Reports of splicing results from a further 7 variants differed in the number of aberrant bands found in each study.…”

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confidence: 99%

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

et al. 2014

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Background Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. Methods We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. Results PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1g>t Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). Conclusions We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

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“…Three BRCA2 recurrent mutations were identified (c.2808_2811delACAA, c.3264dupT and c.9026_9030delATCAT), representing 21.3 % of the total mutations (Table 4). These recurrent mutations have also been found in ICs and most of them have already been reported in studies carried out in Spanish population [26][27][28][29][34][35][36][37][38] or included in mutation databases [30,31] (Table 4). …”

Section: Discussionmentioning

confidence: 91%

BRCA1 and BRCA2 mutations in males with familial breast and ovarian cancer syndrome. Results of a Spanish multicenter study

et al. 2015

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Male breast cancer (MBC) is a rare disease that represents <1% of all breast cancers (BCs). We analyze the results of a multicenter study performed in Spanish familial MBC including family history of hereditary breast and ovarian cancer syndrome (HBOCS) and clinicopathological features. We also study the relationship between BRCA1/BRCA2 mutational status in male relatives affected with cancer (MAC) and, family history and tumor types. The study included 312 men index cases with family history of HBOCS and 61 MAC BRCA1/2 mutation-carriers. Family history, histological grade (HG), clinicopathological and immunohistochemistry data were collected. BRCA1/2 mutation analyses were performed by direct sequencing or screening methods and the large rearrangements by multiplex ligation dependent probe amplification. We found 49 mutation-carriers (15.7%), 95.9% with BRCA2 mutations. BRCA2 mutation-carriers were associated with families with at least one MBC and one BC in female (type II; p = 0.05). Strong association were found between the presence of pathogenic mutations in MBCs and the advanced HG (p = 0.003). c.658_659delTG, c.2808_2811delACAA, c.6275_6276delTT and c.9026_9030delATCAT were the most prevalent mutations. In 61 MAC we found 20 mutations in BRCA1 and 41 in BRCA2. For MAC we show that mutational status was differentially associated with family history (p = 0.018) and tumor type, being BRCA2 mutations linked with BC and prostatic cancer (p = 0.018). MBC caused by BRCA1/2 mutations define two types of MBCs. The most frequent caused by BRCA2 mutation linked to type II families and the rarest one attributed to BRCA1 mutation. Tumor associated with MAC suggest that only BRCA2 mutations have to do with a specific type of cancer (BC and prostatic cancer); but the linkage to tumors is questionable for BRCA1 mutations .

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Assessing the RNA effect of 26 DNA variants in the BRCA1 and BRCA2 genes

Cited by 21 publications

References 23 publications

Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations

Comparative In Vitro and In Silico Analyses of Variants in Splicing Regions of BRCA1 and BRCA2 Genes and Characterization of Novel Pathogenic Mutations

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

BRCA1 and BRCA2 mutations in males with familial breast and ovarian cancer syndrome. Results of a Spanish multicenter study

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