Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay

Nepomuceno, Thales C.; Santos, Ana Paula Paes dos; Fernandes, Vanessa C.; Elias, Anna Beatriz Ribeiro; Gomes, Thiago T.; Suarez‐Kurtz, Guilherme; Iversen, Edwin S.; Couch, Fergus J.; Monteiro, Alvaro N.A.; Carvalho, Marcelo A.

doi:10.1038/s41598-022-20500-4

Cited by 2 publications

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References 51 publications

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“…This variant is located in the linker region between the two BRCT repeats of the BRCT domain, and this might explain why this variant affects neither the TA activity nor the capacity to mediate HRR. Previously, this variant has been shown to be functional in a saturation genome editing assay and a TA assay [ 29 , 35 ], and our results are in concordance with previous studies done on variants in the linker region [ 30 ]. However, Pro1749 is a highly conserved amino acid, and the linker region is included in the PM1 criteria in the CanVIG-UK B RCA1 gene-specific guidelines, which states that these regions have a low rate of benign missense variation and that the residue is important for the functional and/or structural properties of BRCA1 [ 20 ].…”

Section: Discussionsupporting

confidence: 92%

“…The variants p.Phe1668Leu, p.Gly1709Arg, and p.Lys1711Gln showed both lower protein expression than the WT protein and had significantly reduced HRR capacity, but exhibited TA capacity comparable to WT BRCA1 and/or benign controls. In concordance with previous studies [ 30 ], this indicates that even significantly reduced BRCA1 expression levels are sufficient to perform TA at similar levels as the WT protein, and that protein expression levels do not necessarily correlate to the level of protein activity.…”

Section: Discussionsupporting

confidence: 91%

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Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays

et al. 2023

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Background Damaging alterations in the BRCA1 gene have been extensively described as one of the main causes of hereditary breast and ovarian cancer (HBOC). BRCA1 alterations can lead to impaired homologous recombination repair (HRR) of double-stranded DNA breaks, a process which involves the RING, BRCT and coiled-coil domains of the BRCA1 protein. In addition, the BRCA1 protein is involved in transcriptional activation (TA) of several genes through its C-terminal BRCT domain. Methods In this study, we have investigated the effect on HRR and TA of 11 rare BRCA1 missense variants classified as variants of uncertain clinical significance (VUS), located within or in close proximity to the BRCT domain, with the aim of generating additional knowledge to guide the correct classification of these variants. The variants were selected from our previous study “BRCA1 Norway”, which is a collection of all BRCA1 variants detected at the four medical genetic departments in Norway. Results All variants, except one, showed a significantly reduced HRR activity compared to the wild type (WT) protein. Two of the variants (p.Ala1708Val and p.Trp1718Ser) also exhibited low TA activity similar to the pathogenic controls. The variant p.Trp1718Ser could be reclassified to likely pathogenic. However, for ten of the variants, the total strength of pathogenic evidence was not sufficient for reclassification according to the CanVIG-UK BRCA1/BRCA2 gene-specific guidelines for variant interpretation. Conclusions When including the newly achieved functional evidence with other available information, one VUS was reclassified to likely pathogenic. Eight of the investigated variants affected only one of the assessed activities of BRCA1, highlighting the importance of comparing results obtained from several functional assays to better understand the consequences of BRCA1 variants on protein function. This is especially important for multifunctional proteins such as BRCA1.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 91%

Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays

et al. 2023

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Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions

Hovland

Mchaina

Høberg-Vetti

et al. 2023

Genes

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The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

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Assessment of small in-frame indels and C-terminal nonsense variants of BRCA1 using a validated functional assay

Cited by 2 publications

References 51 publications

Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays

Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions

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