147 Matching Annotations
  1. Mar 2021
  2. www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
    Functional characterization of 84 PALB2 variants of uncertain significance
    8
    6. shannon_mcnulty 18 Mar 2021
      WT-PALB2 was associated with robust formation of damage-induced RAD51 foci, whereas the four variants were associated with defective foci formation (Fig. 3d, e).

      AssayResult: 27

      AssayResultAssertion: Normal

      StandardDeviation: 14

      ControlType: Normal; wild type PALB2 cDNA

      Approximation: Exact assay result and standard deviation values not reported; values estimated from Figure 3e.

      CGType:FunctionalAssayResult FuncAssay:2 ValidationControl:WildType AssayControl:Normal
    7. shannon_mcnulty 18 Mar 2021
      ImmunofluorescenceLive cell imaging and microirradiation studies of HeLa cells transfected with peYFP-C1-PALB2 WT or variant constructs were carried out with a Leica TCS SP5 II confocal microscope. To monitor the recruitment of YFP-PALB2 to laser-induced DNA damage sites, cells were microirradiated in the nucleus for 200 ms using a 405-nm ultraviolet (UV) laser and imaged every 30 seconds for 15 minutes. Fluorescence intensity of YFP-PALB2 at DNA damage sites relative to an unirradiated nuclear area was quantified (Supplemental Materials). Cyclin A–positive HeLa cells treated with siCtrl and siRNA against PALB2 were complemented with wild-type and mutant FLAG-tagged PALB2 expression constructs, exposed to 2 Gy of γ-IR, incubated for 6 hours, and subjected to immunofluorescence for RAD51 foci. HeLa cells were fixed with 4% (w/v) paraformaldehyde for 10 minutes at room temperature, washed with tris-buffered saline (TBS), and fixed again with ice-cold methanol for 5 minutes at −20 °C. Cells were incubated for 1 hour at room temperature with the anti-RAD51 (1:7000, B-bridge International, 70-001) and anticyclin A (1:400, BD Biosciences, 611268), and incubated for 1 hour at room temperature with the Alexa Fluor 568 goat antirabbit (Invitrogen, A-11011) and Alexa Fluor 647 goat antimouse (Invitrogen, A-21235) secondary antibodies. Z-stack images were acquired on a Leica CTR 6000 microscope and the number of RAD51 foci per cyclin A–positive cells expressing the indicated YFP-PALB2 constructs was scored with Volocity software v6.0.1 (Perkin–Elmer Improvision). Results represent the mean (± SD) of three independent trials (n = 50 cells per condition). HEK293T cells transfected with PALB2 expression constructs were also subjected to immunofluorescence for PALB2 using the monoclonal anti-FLAG M2 antibody (Sigma) and the Alexa Fluor 568 goat antimouse (Life Technologies) secondary antibody.

      AssayGeneralClass: BAO:0000450 fluorescence microscopy

      AssayMaterialUsed: CLO:0003684 HeLa cell

      AssayDescription: HeLa cells were treated with PALB2 siRNA and transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector), followed by exposure to 2 Gy of γ-IR. Six hours after irradiation, cells were subjected to immunofluorescence for RAD51 foci (where foci formation serves as marker of normal DNA damage repair function).

      AssayReadOutDescription: The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs.

      AssayRange: foci/cell

      AssayNormalRange: Not reported

      AssayAbnormalRange: Not reported

      AssayIndeterminateRange: Not reported

      ValidationControlPathogenic: 0

      ValidationControlBenign: 0

      Replication: Three independent experiments with 50 cells per condition

      StatisticalAnalysisDescription: Kruskal–Wallis test with Dunn's multiple comparison post-test

      CGType:FunctionalAssay FuncAssay:2 BAO:0000450 CLO:0003684 MONDO:0016419 HGNC:26144 AssayRangeType:Quantitative
    8. shannon_mcnulty 18 Mar 2021
      Results for individual PALB2 variants were normalized relative to WT-PALB2 and the p.Tyr551ter (p.Y551X) truncating variant on a 1:5 scale with the fold change in GFP-positive cells for WT set at 5.0 and fold change GFP-positive cells for p.Y551X set at 1.0. The p.L24S (c.71T>C), p.L35P (c.104T>C), p.I944N (c.2831T>A), and p.L1070P (c.3209T>C) variants and all protein-truncating frame-shift and deletion variants tested were deficient in HDR activity, with normalized fold change <2.0 (approximately 40% activity) (Fig. 1a).

      AssayResult: 5.3

      AssayResultAssertion: Normal

      StandardErrorMean: 0.46

      CGType:FunctionalAssayResult CG:BulkAnnotation FuncAssay:1 Variant:2 CAID:CA151212 ClinVarID:126582 ValidationControl:Benign
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    ncbi.nlm.nih.gov/pmc/articles/PMC7056643/
  3. www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
    A global functional analysis of missense mutations reveals two major hotspots in the PALB2 tumor suppressor
    23
    1. shannon_mcnulty 18 Mar 2021
      RAD51 foci assayHeLa cells were seeded on glass coverslips in 6-well plates at 225 000 cells per well. Knockdown of PALB2 was performed 18 h later with 50 nM PALB2 siRNA using Lipofectamine RNAiMAX (Invitrogen). After 5 h, cells were subjected to double thymidine block. Briefly, cells were treated with 2 mM thymidine for 18 h and release into fresh media for 9 h. Complementation using 800 ng of the peYFP-C1 empty vector or the indicated siRNA-resistant YFP-PALB2 construct was carried out with Lipofectamine 2000 during that release time. Then, cells were treated with 2 mM thymidine for 17 h and protected from light from this point on. After 2 h of release from the second block, cells were irradiated with 2 Gy and processed for immunofluorescence 4 h post-irradiation. Unless otherwise stated, all immunofluorescence dilutions were prepared in PBS and incubations performed at room temperature with intervening washes in PBS. Cell fixation was carried out by incubation with 4% paraformaldehyde for 10 min followed by 100% ice-cold methanol for 5 min at −20°C. This was succeeded by permeabilization in 0.2% Triton X-100 for 5 min and a quenching step using 0.1% sodium borohydride for 5 min. After blocking for 1 h in a solution containing 10% goat serum and 1% BSA, cells were incubated for 1 h with primary antibodies anti-RAD51 (1 :7000, B-bridge International, #70–001) and anti-cyclin A (1:400, BD Biosciences, #611268) diluted in 1% BSA. Secondary antibodies Alexa Fluor 568 goat anti-rabbit (Invitrogen, #A-11011) and Alexa Fluor 647 goat anti-mouse (Invitrogen, #A-21235) were diluted 1:1000 in 1% BSA and applied for 1 h. Nuclei were stained for 10 min with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) prior to mounting onto slides with 90% glycerol containing 1 mg/ml paraphenylenediamine anti-fade reagent. Z-stack images were acquired on a Leica CTR 6000 microscope using a 63× oil immersion objective, then deconvolved and analyzed for RAD51 foci formation with Volocity software v6.0.1 (Perkin-Elmer Improvision). The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored using automatic spot counting by Volocity software and validated manually. Data from three independent trials (total n = 225 cells per condition) were analyzed for outliers using the ROUT method (Q = 1.0%) in GraphPad Prism v6.0 and the remaining were reported in a scatter dot plot. Intensity values, also provided by Volocity, of 500 RAD51 foci from a representative trial were normalized to the WT mean and reported in a scatter dot plot. Horizontal lines on the plots designate the mean values.

      AssayGeneralClass: BAO:0000450 fluorescence microscopy

      AssayMaterialUsed: CLO:0003684 HeLa cell

      AssayDescription: HeLa cells were treated with PALB2 siRNA and synchronized to G1/S phase by double thymidine block. Cells were then transfected with peYFP-PALB2 expressing PALB2 variants (or empty vector) and irradiated with 2 Gy. Four hours after irradiation, cells were subjected to immunofluorescence for RAD51 foci (where foci formation serves as marker of normal DNA damage repair function).

      AssayReadOutDescription: The number of RAD51 foci per cyclin A-positive cells expressing the indicated YFP-PALB2 constructs was scored and presented as percentage change relative to the wild type mean RAD51 foci number per cell.

      AssayRange: %

      AssayNormalRange: Not reported

      AssayAbnormalRange: Not reported

      AssayIndeterminateRange: Not reported

      ValidationControlPathogenic: 1

      ValidationControlBenign: 3

      Replication: Three independent experiments, each with 225 cells per condition

      StatisticalAnalysisDescription: Kruskal–Wallis test with Dunn's multiple comparison post-test

      CGType:FunctionalAssay FuncAssay:2 BAO:0000450 CLO:0003684 MONDO:0016419 HGNC:26144 AssayRangeType:Quantitative UO:0000187
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    ncbi.nlm.nih.gov/pmc/articles/PMC6847799/
  4. www.nature.com www.nature.com
    Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2
    79
    1. shannon_mcnulty 18 Mar 2021
      sensitivity to PARPi treatment using a cellular proliferation assay

      AssayGeneralClass: BAO:0002805 cell proliferation assay

      AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line

      AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; exposure to PARP inhibitor Olaparib for 48 h inhibits end-joining mediated by PARP and sensitizes cells to DNA damage; cell survival is measured by FACS 24 h after Olaparib washout

      AssayReadOutDescription: Relative resistance to PARPi represented as cell survival relative to wild type, which was set to 100%

      AssayRange: %

      AssayNormalRange: PARPi resistance levels comparable to that of cells expressing wild type PALB2; no numeric threshold given

      AssayAbnormalRange: PARPi resistance levels ≤30% of wild type

      AssayIndeterminateRange: Not reported

      ValidationControlPathogenic: 12

      ValidationControlBenign: 9

      Replication: 2 independent experiments

      StatisticalAnalysisDescription: Not reported

      CGType:FunctionalAssay FuncAssay:2 BAO:0002805 CLO:0037317 MONDO:0016419 HGNC:26144 AssayRangeType:Quantitative UO:0000187
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    nature.com/articles/s41467-019-13194-2
  5. www.cell.com www.cell.com
    High-Throughput Reclassification of SCN5A Variants
    1
    1. shannon_mcnulty 18 Mar 2021
      Most Suspected Brugada Syndrome Variants Had (Partial) Loss of Function

      AssayResult: 113.2

      AssayResultAssertion: Normal

      ReplicateCount: 30

      StandardErrorMean: 13.9

      Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

      CGType:FunctionalAssayResult CG:BulkAnnotation FuncAssay:1 Variant:2 CAID:CA352149766
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    cell.com/ajhg/fulltext/S0002-9297(20)30162-2
  6. jmg.bmj.com jmg.bmj.com
    Blood functional assay for rapid clinical interpretation of germline TP53 variants
    1
    1. dkanavy 18 Mar 2021
      This new quantitative assay, based on both RT-QMPSF and RT-MLPA, was first validated on 31 lymphoblastoid cell lines derived from patients with LFS harbouring different germline heterozygous TP53 variants

      AssayGeneralClass: BAO:0010044 targeted transcriptional assay

      AssayMaterialUsed: BTO:0000773 lymphoblastoid cell line derived from control individuals or individuals with germline TP53 variants

      AssayDescription: Comparative transcriptomic analysis using RNA-Seq to compare EBV cell lines of wild type and pathogenic TP53 in the context of genotoxic stress induced by doxorubicin treatment. p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for the three wild-type TP53 individuals.

      AdditionalDocument: PMID: 23172776

      AssayReadOutDescription: The p53 mRNA levels were expressed as a ratio of the normal values obtained for 3 TP53 wild-type control individuals.

      AssayRange: UO:0000187 the p53 RNA levels were evaluated and expressed as a percentage of the mean levels obtained for three wild-type TP53 individuals.

      AssayNormalRange: N/A

      AssayAbnormalRange: N/A

      AssayIndeterminateRange: N/A

      AssayNormalControl: wild type TP53

      AssayAbnormalControl: LFS patient cells

      ValidationControlPathogenic: 8 Individuals with dominant-negative TP53 missense variants, 10 Individuals with null TP53 variants, and 13 Individuals with other TP53 missense variants

      ValidationControlBenign: 3 patients with wild type TP53

      Replication: experiments were performed in triplicates.

      StatisticalAnalysisDescription: Differentially expressed genes between doxorubicin-treated and untreated cells were arbitrarily defined using, as filters, a P<0.01 and fold-change cutoffs >2 or <2, for up and down regulation, respectively. The resultant signal information was analyzed using one-way analysis of variance (ANOVA, P= 0.001), assuming normality but not equal variances with a Benjamani–Hochberg correction for multiple comparisons using three groups: controls, null, and missense mutations.

      SignificanceThreshold: P=0.001

      Comment: statistical analysis and P value from previous publication.

      FuncAssay:2 BAO:0010044 BTO:0000773 MONDO:0018875 HGNC:11998 UO:0000187 AssayReadOutType:Quantitative CGType:FunctionalAssay
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    jmg.bmj.com/content/early/2020/10/13/jmedgenet-2020-107059
  7. Feb 2021
  8. jmg.bmj.com jmg.bmj.com
    Blood functional assay for rapid clinical interpretation of germline TP53 variants
    35
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    jmg.bmj.com/content/early/2020/10/13/jmedgenet-2020-107059