Probing Structure-Function Relationships in Missense Variants in the Carboxy-Terminal Region of BRCA1

Carvalho, Renato S.; Abreu, Renata B. V.; Velkova, Aneliya; Marsillac, Sylvia M.; Rodarte, Renato Santos; Suarez‐Kurtz, Guilherme; Iversen, Edwin S.; Monteiro, Alvaro N.A.; Carvalho, Marcelo A.

doi:10.1371/journal.pone.0097766

Cited by 8 publications

(7 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1B). This missense mutation has been previously reported as a pathogenic mutation, predisposing carriers to breast and ovarian cancer (13–15). There were no other compelling variants that explained her developmental phenotype, and none that were shared with other patients diagnosed with Dubowitz syndrome.…”

Section: Resultsmentioning

confidence: 91%

Biallelic Mutations in BRCA1 Cause a New Fanconi Anemia Subtype

et al. 2015

View full text Add to dashboard Cite

Deficiency in BRCA dependent DNA inter-strand crosslink (ICL) repair is intimately connected to breast cancer susceptibility and to the rare developmental syndrome, Fanconi Anemia (FA). Bona fide FA proteins, BRCA2 (FANCD1), PALB2 (FANCN), and BRIP1 (FANCJ) interact with BRCA1 during ICL repair. However, lack of detailed phenotypic and cellular characterization of a patient with biallelic BRCA1 mutations has precluded assignment of BRCA1 as a definitive FA susceptibility gene. Here we report the presence of biallelic BRCA1 mutations in a woman with multiple congenital anomalies consistent with a FA-like disorder and breast cancer at age 23. Patient cells exhibited deficiency in BRCA1 (FANCS) and Rad51 localization to DNA damage sites, combined with radial chromosome formation and hypersensitivity to ICL inducing agents. Restoration of these functions was achieved by ectopic introduction of a BRCA1 transgene. These observations provide evidence in support of BRCA1 as a new Fanconi anemia subtype (FA-S).

show abstract

Section: Resultsmentioning

confidence: 91%

Biallelic Mutations in BRCA1 Cause a New Fanconi Anemia Subtype

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Two of the variants chosen for retest analysis were the C1787S and G1788D. The C1787S variant previously scored as non-pathogenic, 13 but was classified as IARC 5 using the genetic data. 14 As it was always seen in conjunction with G1788D (likely in cis ), 14 we tested these variants together and separately.…”

Section: Resultsmentioning

confidence: 99%

“…All new and retested variants were analysed using the TA luciferase reporter assay as previously described. 13 Briefly, BRCA1 constructs were co-transfected in HEK293FT cells with the pG5Luc plasmid, encoding a Luciferase reporter gene driven by GAL4 binding sites, and the phGR-TK plasmid, which constitutively expresses the internal control Renilla luciferase. Transcriptional activity was assayed with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) 24 h after transfection.…”

Section: Methodsmentioning

confidence: 99%

Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

et al. 2016

View full text Add to dashboard Cite

Variants of Uncertain Significance (VUS) are genetic variants whose association with a disease phenotype has not been established. They are a common finding in sequencing-based genetic tests and pose a significant clinical challenge. The objective of this study was to assess the use of functional data to classify variants according to pathogenicity. We conduct functional analysis of a large set of BRCA1 VUS combining a validated functional assay with VarCall, a Bayesian hierarchical model to estimate the likelihood of pathogenicity given the functional data. The results from the functional assays were incorporated into a joint analysis of 214 BRCA1 VUS to predict their likelihood of pathogenicity (breast cancer). We show that applying the VarCall model (1.0 sensitivity; lower bound of 95% confidence interval (CI) = 0.75 and 1.0 specificity; lower bound of 95% CI = 0.83) to the current set of BRCA1 variants, use of the functional data would significantly reduce the number of VUS associated with the C-terminal region of the BRCA1 protein by ~ 87%. We extend this work developing yeast-based functional assays for two other genes coding for BRCT domain containing proteins, MCPH1 and MDC1. Analysis of missense variants in MCPH1 and MDC1 shows that structural inference based on the BRCA1 data set can aid in prioritising variants for further analysis. Taken together our results indicate that systematic functional assays can provide a robust tool to aid in clinical annotation of VUS. We propose that well-validated functional assays could be used for clinical annotation even in the absence of additional sources of evidence.

show abstract

“…Both lines are derived from ascites obtained from HGSOC patients and harbor p53 mutations (A13-12-12: splice site mutation c.782 + 1G > A; OVCAR3: p.Arg248Gln) with A13-2-12 showing an additional BRCA1 mutation (p.Ala1752Thr). This mutation is reportedly pathogenic [ 63 ].…”

Section: Resultsmentioning

confidence: 99%

RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis

Heinze

Hölzer²,

Ungelenk

et al. 2021

Cancers

View full text Add to dashboard Cite

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis.

show abstract

Probing Structure-Function Relationships in Missense Variants in the Carboxy-Terminal Region of BRCA1

Cited by 8 publications

References 33 publications

Biallelic Mutations in BRCA1 Cause a New Fanconi Anemia Subtype

Biallelic Mutations in BRCA1 Cause a New Fanconi Anemia Subtype

Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance

RUNX3 Transcript Variants Have Distinct Roles in Ovarian Carcinoma and Differently Influence Platinum Sensitivity and Angiogenesis

Contact Info

Product

Resources

About