Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition

Santos, E. Santana dos; Caputo, Sandrine M.; Castéra, Laurent; Gendrot, M.; Briaux, Adrien; Breault, Magali; Krieger, Sophie; Rogan, Peter K.; Mucaki, Eliseos J.; Burke, Les J.; Bièche, Ivan; Houdayer, Claude; Vaur, Dominique; Stoppa‐Lyonnet, Dominique; Brown, Melissa A.; Lallemand, François; Rouleau, Étienne

doi:10.1007/s10549-017-4602-0

Cited by 19 publications

(23 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is hypothesized that variants in these regions have potential to modulate gene expression (Stranger et al., ; Stranger et al., ) and impact on relative disease risk, possibly in collaboration with multiple other low‐, moderate‐, and high‐risk variants (Manolio et al., ). This extends and validates our previous study (Santana dos Santos et al., ) by using a larger number patients analyzed over nine geographical locations, identifying additional BC‐associated variants, and showing that a subset of these variants modulate binding of specific TFs. Further, we have compared results from our bioinformatics and functional analysis to variant classifications based on ENIGMA BRCA1/2 guidelines for high‐risk variation in these genes.…”

Section: Discussionsupporting

confidence: 89%

“…This may reflect the differential availability of crucial TFs in MDA‐MB‐468 cells (Kao et al., ) and highlights the importance of undertaking that assays for functional activity of variants in more than one cell line. Three variants, BRCA1 :c.‐380G>A, BRCA2 :c.‐296C>T, and BRCA2 :c.‐218G>A, were also analyzed in our earlier paper (Santana dos Santos et al., ). Although the cell lines used in the two studies were different (MDA‐MB‐231 in Santana dos Santos et al., and MCF7 and MDA‐MB‐468 here), the trends are the same in five out of six analyses.…”

Section: Discussionmentioning

confidence: 99%

“…Three variants, BRCA1 :c.‐380G>A, BRCA2 :c.‐296C>T, and BRCA2 :c.‐218G>A, were also analyzed in our earlier paper (Santana dos Santos et al., ). Although the cell lines used in the two studies were different (MDA‐MB‐231 in Santana dos Santos et al., and MCF7 and MDA‐MB‐468 here), the trends are the same in five out of six analyses. The difference for BRCA2 :c.‐296C>T, which causes a significant decrease in MDA‐MB‐231 and MCF7 cells, but not MDA‐MB‐468 cells, may again be indicative of differential gene expression in BC cell lines (Kao et al., ).…”

Section: Discussionmentioning

confidence: 99%

“…Gene expression is controlled at many levels with key regulatory elements being housed in noncoding regions of the genome, such as gene promoters, introns, long‐range elements, and 5′ and 3′ UTRs. The BRCA1 gene is regulated at the transcriptional and posttranscriptional levels, with functional proximal and distal regulatory elements being described in the promoter, introns, and UTRs, by us and others (Brewster et al., ; Brown et al., ; Santana dos Santos et al., ; Saunus et al., ; Tan‐Wong, French, Proudfoot, & Brown, ; Wardrop, Brown, & kConFab, ; Wiedemeyer, Beach, & Karlan, ). Although less studied, the BRCA2 promoter has also been mapped and characterized (reviewed in Wiedemeyer et al., ).…”

Section: Introductionmentioning

confidence: 96%

See 3 more Smart Citations

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

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 96%

See 2 more Smart Citations

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…A recent study analysing the functional impact of non-coding BRCA1 variants remarked the importance of this intron because it contains regulatory regions that may affect BRCA1 promoter activity 33. In our study, we could not formally exclude that this variant contributes to the allelic imbalance observed in the carrier.…”

Section: Discussionmentioning

confidence: 73%

Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer

Montalban¹,

Bonache²,

Moles‐Fernández³

et al. 2018

J Med Genet

View full text Add to dashboard Cite

BackgroundGenetic analysis of BRCA1 and BRCA2 for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility.MethodsWe analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 during conventional analysis. Rare variants (MAF <0.005) predicted to create/activate splice sites were selected for further characterisation in patient RNA. The splicing outcome was analysed by RT-PCR and Sanger sequencing, and allelic imbalance was also determined when heterozygous exonic loci were present.ResultsA novel transcript was detected in BRCA1 c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population.ConclusionScreening of BRCA1/2 intronic regions has identified the first BRCA1 deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.

show abstract

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

View full text Add to dashboard Cite

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.

show abstract

Assessment of the functional impact of germline BRCA1/2 variants located in non-coding regions in families with breast and/or ovarian cancer predisposition

Abstract: In this study, we highlighted some variants among many, modulating negatively the promoter activity of BRCA1 or 2 and thus having a potential impact on the risk of developing cancer. This selection makes it possible to conduct future validation studies on a limited number of variants.

Cited by 19 publications

References 40 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

Screening of BRCA1/2 deep intronic regions by targeted gene sequencing identifies the first germline BRCA1 variant causing pseudoexon activation in a patient with breast/ovarian cancer

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

Contact Info

Product

Resources

About