Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants

Fraile-Bethencourt, Eugenia; Valenzuela-Palomo, Alberto; Díez-Gómez, Beatriz; Infante, Mar; Durán, Mercedes; Marcos, Germán; Lastra, Enrique; Gómez-Barrero, Susana; Velasco, Eladio A.

doi:10.1007/s10549-018-4826-7

Cited by 9 publications

(12 citation statements)

References 45 publications

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“…As TFs can function as activators or repressors, a variant‐associated change in TF binding can result in either a decrease or an increase in promoter (or other regulatory element) activity. Differences in the quanta and direction of promoter activity have been reported previously (e.g., Fraile‐Bethencourt et al., ; Santana dos Santos et al., ) and have also been shown to differ between cell lines potentially reflecting the availability of TFs or cofactors (e.g., Zn).…”

Section: Discussionsupporting

confidence: 58%

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

et al. 2018

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The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early‐onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.‐287C>T and PAX5 binding to BRCA2:c.‐296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

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Section: Discussionsupporting

confidence: 58%

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

et al. 2018

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“…Variants of disease-genes are typically assessed according to their predicted impact on protein translation, so protein truncating variants (frameshift and nonsense) are usually classified as damaging variants. However, variants might also have an impact on RNA expression and, e.g., disrupt transcription initiation, miRNA regulation, or splicing [23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

Sanoguera-Miralles

Valenzuela-Palomo

Bueno-Martínez

et al. 2020

Cancers

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Hereditary breast and/or ovarian cancer is a highly heterogeneous disease with more than 10 known disease-associated genes. In the framework of the BRIDGES project (Breast Cancer Risk after Diagnostic Gene Sequencing), the RAD51C gene has been sequenced in 60,466 breast cancer patients and 53,461 controls. We aimed at functionally characterizing all the identified genetic variants that are predicted to disrupt the splicing process. Forty RAD51C variants of the intron-exon boundaries were bioinformatically analyzed, 20 of which were selected for splicing functional assays. To test them, a splicing reporter minigene with exons 2 to 8 was designed and constructed. This minigene generated a full-length transcript of the expected size (1062 nucleotides), sequence, and structure (Vector exon V1- RAD51C exons_2-8- Vector exon V2). The 20 candidate variants were genetically engineered into the wild type minigene and functionally assayed in MCF-7 cells. Nineteen variants (95%) impaired splicing, while 18 of them produced severe splicing anomalies. At least 35 transcripts were generated by the mutant minigenes: 16 protein-truncating, 6 in-frame, and 13 minor uncharacterized isoforms. According to ACMG/AMP-based standards, 15 variants could be classified as pathogenic or likely pathogenic variants: c.404G > A, c.405-6T > A, c.571 + 4A > G, c.571 + 5G > A, c.572-1G > T, c.705G > T, c.706-2A > C, c.706-2A > G, c.837 + 2T > C, c.905-3C > G, c.905-2A > C, c.905-2_905-1del, c.965 + 5G > A, c.1026 + 5_1026 + 7del, and c.1026 + 5G > T.

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“…Variants are usually classified attending to their impact on the protein translated, but disruption of other gene expression steps may occur, including transcription initiation, miRNA and long noncoding RNA regulation, RNA stability or post‐transcriptional modifications . Splicing is a gene expression step whereby introns are excised and exons are joined sequentially.…”

Section: Introductionmentioning

confidence: 99%

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

Fraile-Bethencourt

Valenzuela-Palomo

Díez-Gómez

et al. 2019

The Journal of Pathology

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Splicing disruption is a common mechanism of gene inactivation associated with germline variants of susceptibility genes. To study the role of BRCA2 mis-splicing in hereditary breast/ovarian cancer (HBOC), we performed a comprehensive analysis of variants from BRCA2 exons 2-9, as well as the initial characterization of the regulatory mechanisms of such exons. A pSAD-based minigene with exons 2-9 was constructed and validated in MCF-7 cells, producing the expected transcript (1016-nt/V1-BRCA2_exons_2-9-V2). DNA variants from mutational databases were analyzed by NNSplice and Human Splicing Finder softwares. To refine ESE-variant prediction, we mapped the regulatory regions through a functional strategy whereby 26 exonic microdeletions were introduced into the minigene and tested in MCF-7 cells. Thus, we identified nine spliceogenic ESE-rich intervals where ESE-variants may be located. Combining bioinformatics and microdeletion assays, 83 variants were selected and genetically engineered in the minigene. Fifty-three changes impaired splicing: 28 variants disrupted the canonical sites, four created new ones, 10 abrogated enhancers, eight created silencers and three caused a double-effect. Notably, nine spliceogenic-ESE variants were located within ESE-containing intervals. Capillary electrophoresis and sequencing revealed more than 23 aberrant transcripts, where exon skipping was the most common event. Interestingly, variant c.67G>A triggered the usage of a noncanonical GC-donor 4-nt upstream. Thirty-six variants that induced severe anomalies (>60% aberrant transcripts) were analyzed according to the ACMG guidelines. Thus, 28 variants were classified as pathogenic, five as likely pathogenic and three as variants of uncertain significance. Interestingly, 13 VUS were reclassified as pathogenic or likely pathogenic variants. In conclusion, a large fraction of BRCA2 variants (∼64%) provoked splicing anomalies lending further support to the high prevalence of this disease-mechanism. The low accuracy of ESE-prediction algorithms may be circumvented by functional ESE-mapping that represents an optimal strategy to identify spliceogenic ESE-variants. Finally, systematic functional assays by minigenes depict a valuable tool for the initial characterization of splicing anomalies and the clinical interpretation of variants.

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Genetic dissection of the BRCA2 promoter and transcriptional impact of DNA variants

Cited by 9 publications

References 45 publications

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

Mis‐splicing in breast cancer: identification of pathogenic BRCA2 variants by systematic minigene assays

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