| A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U | V | W | X | Y | Z | AA | ||
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1 | Gene | Reference | PMID | Evidence Weighting | Please see main CanVIG-UK consensus specification (https://www.cangene-canvaruk.org/canvig-uk-guidance) and gene specific guidance (https://www.cangene-canvaruk.org/gene-specific-recommendations) for recommendations on combining the results of multiple assays and interpreting conflicting results | CanVar datasource | Classified variant controls | Prior Probability (P1) | Assay result | "+1 proportions" Posterior Probability (P2)¥ | OddsPath = [P2 x (1-P1)] / [(1-P2) x P1] | ¥ Note: this estimate incorporates fully discordant results, as well as inderminate results and adds 1.0 to both the numerator and denominator for the benignity calculations as the "+1" in the formula in the original Brnich et al, 2020 paper led to a lack of conservatism | CanVIG-UK member reviewing | CanVIG-UK reviewer centre | CanGene-CanVar team checked? | |||||||||||||
2 | PS3 | BS3 | Additional CanVIG-UK recommendations | Assay scoring | Threshold functionally normal | Threshold functionally abnormal | Total | Pathogenic | Benign | Functionally abnormal | Indeterminate | Functionally normal | Pathogenic | Benign | Pathogenic | Benign | Comment: assessment according to Brnich et al 2020 | |||||||||||
3 | Pathogenic controls | Benign controls | Pathogenic controls | Benign controls | ||||||||||||||||||||||||
4 | BRCA1 | Bouwman et al, 2020 | 32546644 | Strong | Strong | Bouwman et al, 2020 consists of 3 constituent assays to be scored as 1 assay. | P = the posterior probability for a variant to be deleterious based on its mean-normalised IC50 (olaparib and cisplatin sensitivity assays) or GFP percentage (DR-GFP assay) over 3 biological replicates. | 0.01 < P ≤ 0.05: likely neutral P ≤ 0.01: neutral 3 constituent assays to be scored as 1 assay, allowing for 1 assay intermediate outcome- i.e. all assays (likely) neutral, or 2 assays (likely) neutral and 1 assay intermediate. “Not clear” refers to opposite categorisation ± the standard deviation of repeat experiments and should be treated as conflicting assay results (i.e. do not use as evidence towards BS3). | P > 0.99: deleterious 0.95 < P ≤ 0.99: likely deleterious 3 constituent assays to be scored as 1 assay, allowing for 1 assay intermediate outcome- i.e. all assays (likely) deleterious, or 2 assays (likely) deleterious and 1 assay intermediate. “Not clear” refers to opposite categorisation ± the standard deviation of repeat experiments and should be treated as conflicting assay results (i.e. do not use as evidence towards PS3). | Supplementary table S1_S2 | 50 | 25 | 25 | 0.50 | 25 | 0 | 0 | 0 | 25 | 0.96 | 0.04 | 25.00 | 0.04 | Numeric assessment (OddsPath) | ||||
5 | BRCA1 | Findlay et al, 2018 | 30209399 | Strong | Strong | HAP1 functional score = cell survival scores, log- normalised across experiments using global medians. | HAP1 functional score >-0.748 | HAP1 functional score <-1.328 | Supplementary table 1 | 188 | 167 | 21 | 0.89 | 162 | 0 | 6 | 0 | 20 | 0.99 | 0.05 | 20.37 | 0.01 | Numeric assessment (OddsPath) | |||||
6 | BRCA1 | Fernandes et al, 2019 | 30765603 | Supporting | Supporting | Percentage mean of wild type transcriptional activity. Two approaches for transcription data normalisation: 1) vector only expression levels 2) vector and ectopic protein expression levels N.B. 35 variants, all located within the BRCT domain, showed discordant results between the two normalisation methods | Mean ≥80% wild type activity | Mean <80% wild type activity | Supplementary data not provided in extractable format for CanVar. See figures 2-4 in paper | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Basic controls included, variants assessed using 3 technical replicates for each variant in at least 2 independent experiments for a total of a least 6 measurements per variant. Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). Assigned PS3/BS3_sup on basis of assay having been "broadly accepted historically" | |||||
7 | BRCA1 | Petitalot et al, 2019 | 30257991 | Supporting | Supporting | HR assessed by frequency of GFP positive cells normalised to wild type BRCA1. Statistical significance (HR efficiency significantly different to wild type) presented by p values. P= statistical significance upon comparison of frequency of GFP positive cells in variant transfected (normalised to WT) compared to wild type control. Designated classification (1P, 2P, 3P) based on combining assay results: Localisation assay: immunofluorescence to show BRCA1 localisation following Mitomycin treatment. Assessed by percentage fluorescence in the nucleus/total in the cell and statistical significance determined by two tailed Student t test. Solubility assay: separation of soluble and insoluble fractions for gel analysis. Phosphopeptide-binding assay: isothermal titration calorimetry and size-exclusion chromatography | HR assay: P ≥0.05 Thresholds not evident for other assays | HR assay: P <0.05 Thresholds not evident for other assays | HR: supplementary data, sheet 2 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Basic controls included, each variant tested in triplicate in at least 4 independent experiments. 7 'causal' and 6 'neutral' variants selected as controls on basis of BRCAshare classifications (different ClinVar classifications presented in supplementary table 1) Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). Assigned PS3/BS2_sup on basis of assay having been "broadly accepted historically" | |||||
8 | BRCA1 | Starita et al, 2018 | 30219179 | Strong | Moderate | Ratio of variant frequency in GFP+ cells to GFP- cells, normalised to equivalently calculated for wild type transgene. To compare variants to each other, generated a binary classifier ("depleted" vs "not depleted") comparing the significance of each variant's depletion in test versus control experiments at the same read count in the same experiment. Assays are scored according to the number of replicates (out of 3 or 4) which are classed as depleted or not depleted for a given variant | Zero replicates showing depletion | 3-4 replicates showing depletion | Table S7 (column countDepleted)- table uses protein coding nomenclature only | 46 | 12 | 34 | 0.26 | 11 | 0 | 0 | 1 | 34 | 0.92 | 0.06 | 31.17 | 0.17 | Numeric assessment (OddsPath): ClinVar (likely) benign and (likely) pathogenic controls presented in Table S2. Two variants excluded from Brnich assessment due to unclear coding nomenclature. (12 rather than 14 pathogenic variants). Controls previously tested using the same assay. Quadruplicate assessment of variant libraries. Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). | |||||
9 | BRCA2 | Biswas et al., 2020 | 33293522 | Supporting | Supporting | Please see ENIGMA VCEP Group guidance, Supplementary Table 9, for per-variant assessment of evidence use (including the Biswas et al. 2020 assay) | Complementation of BRCA2 function by transgene constructs in BRCA2-null mouse embronic stem (mES) cells evaluated by quantification of cell survival via cell number. Where sufficient cells survive, they are evaluated for sensitivity against 6 to agents Categorical functionality groupings depending on a probability of impact function value derived from the VarCall computational model; assigned as functional (F), indeterminate (I) or non-functional (NF) | PIF ≤ 0.05 (F) | P > 0.99 (NF) | Table 1 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Basic controls included, each variant tested in triplicate in at least 2 independent experiments. 5 pathogenic and 5 benign variants selected as controls on basis of ClinVar and BRCA Exchange classifications (one pathogenic missense control ostensibly selected on the basis of Align-GVGD prediction). Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). Assigned PS3_sup/BS3_sup on basis of assay having been "broadly accepted historically" | Charlie Rowlands | ICR | ||
10 | BRCA2 | Guidugli et al, 2018 | 29394989 | Strong | Moderate | HDR assay score = HDR-fold change (assessed using GFP expressing cell fold changes) normalised and rescaled in relation to a pathogenic and wild type control. | HDR assay score >2.25 | HDR assay score <1.78 | Table 1 in paper | 34 | 13 | 21 | 0.38 | 13 | 0 | 0 | 0 | 21 | 0.93 | 0.05 | 21 | 0.08 | Numeric assessment (OddsPath) | |||||
11 | BRCA2 | Hart et al, 2019 | 29884841 | Strong | Moderate | Data presented in Table 1 for BRCA2 HDR activity to be used for PS3/BS3 application. | FC = fold change in GFP positive cells | FC = >2.41 | FC = <1.66 | Table 1 in paper | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | No additional controls evident. Evidence weightings applied based on validation from previous iterations of the assay by the Couch lab (Guidugli et al, 2018) Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). | ||||
12 | BRCA2 | Hu et al., 2022 | 35736817 | Strong | Strong | Apply at STR | DBD domain BRCA2 missense variants assayed, and GFP expression assessed for each transfected cell with the BRCA2 missense variant (where successful rescue of HDR pathway resulted in GFP expression) | HDR score of >2.50 | HDR score of <1.49 | Table 1, Table 2 | 56 | 21 | 35 | 0.375 | 21 | 0 | 0 | 0 | 35 | 0.954545 | 0.027778 | 35 | 0.047619 | Set of 21 known P/LP and 35 known B/LB variants, none predicted to cause splicing effect by SpliceAI. Assessment of OddsPath showing PS3 = STR and BS3 = STR | Sophie Allen | ICR | ||
13 | BRCA2 | Hu et al., 2024 | 38417439 | Strong | Strong | Apply at STR | DBD domain BRCA2 missense variants assayed, and GFP expression assessed for each transfected cell with the BRCA2 missense variant (where successful rescue of HDR pathway resulted in GFP expression) | HDR score of >2.50 | HDR score of <1.49 | Table S1 | 56 | 21 | 35 | 0.38 | 21 | 0 | 0 | 0 | 35 | 0.95 | 0.03 | 35.00 | 0.05 | Set of 21 known P/LP and 35 known B/LB variants, none predicted to cause splicing effect by SpliceAI. Assessment of OddsPath showing PS3 = STR and BS3 = STR | Charlie Rowlands | ICR | ||
14 | BRCA2 | Mesman et al, 2019 | 29988080 | Moderate | Moderate | Evidence weighting "moderate" applies to complement assay only due to insufficient pathogenic controls for validation of HR and cisplatin sensitivity assays. All variants assessed by Mesman et al have been included in the complementation assay. | Complementation by BRCA2 variants of the cell lethal phenotype imposed by Cre-mediated loss of Brca2 was visualized by methylene blue staining of arising HAT-resistant clones. HDR capacity, as measured by the repair of an I-Sce1 induced double strand break (DSB) in the direct repeat green fluorescent protein (DR-GFP) reporter, relative to the HDR levels observed in wild type (WT) BRCA2-expressing cells. Cisplatin sensitivity of BRCA2 variants normalized to the average sensitivity of WT BRCA2-expressing cells (IC50 values relative to the IC50 of WT controls presented in percentages) | Complementation: 'Yes' | Complementation: 'No' | Table 2 in paper | 35 | 15 | 20 | 0.43 | 14 | 0 | 0 | 1 | 20 | 0.93 | 0.09 | 18.67 | 0.13 | Numeric assessment (OddsPath). Evidence weighting "moderate" only applies to complement assay due to insufficient pathogenic controls for validation of HR and cisplatin sensitivity assays. All variants assessed by Mesman et al have been included in the complementation assay. Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (ENIGMA). | ||||
15 | BRCA2 | Richardson et al, 2021 | 33609447 | Strong | Strong | HDR assay score = HDR-fold change (assessed using GFP expressing cell fold changes) normalised and rescaled in relation to a pathogenic and wild type control. | HDR assay score >2.25 | HDR assay score <1.78 | Table S1- table uses protein coding nomenclature only | 56 | 19 | 37 | 0.34 | 19 | 0 | 0 | 0 | 37 | 0.95 | 0.03 | 37.00 | 0.05 | Numeric assessment (OddsPath): control variants in Table S1 can be selected under column AD "Standards for HDR Validation". Control variant counts listed here vary to in paper values following removal of non-ClinVar/expert group assigned (likely) pathogenic and (likely) benign classifications. Some of the variants included were used for the original identification of HDR thresholds (Guidugli et al, 2018) as these were indistinguishable from 'novel' control variants listed in column AD, Table S1. HDR score cut-offs stated are at the 95% probability of pathogenicity/benignity for consistency with the Guidugli et al, 2018 thresholds. All variants were analysed in duplicate experiments for at least two independently derived clones. | |||||
16 | BRCA2 | Ikegami et al, 2020 | 32444794 | Strong | Strong | Please see ENIGMA VCEP Group guidance, supplementary table 9, for per-variant assessment of evidence use (including the Ikegami et al 2020 assay) | Combination of log-normalised data from drug sensitivity assays (olaparib, niraparib, rucaparib, carboplatin) used to calculate Bayes Factor for each variant, giving an fClass of 1,2,3,4 or 5 for each assay. | All assays: Bayes Factor ≤ 0.05 (fClass 1 or 2) | All assays: Bayes Factor >18.7 (fClass 4 or 5) | SuppData5 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical group. Use evidence weighting in accordance to the ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel (Supplementary Table 9): https://clinicalgenome.org/affiliation/50087/ | ||||
17 | TP53 | Kato et al, 2003 | 12826609 | ClinGen TP53 Expert Panel Specifications | ClinGen TP53 Expert Panel Specifications | Median score across transactivation assay for 8 promoter sequences. | Median= 76-140% transactivation activity | Median= ≤20% transactivation activity | IARC/ClinGen | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical group. Use evidence weighting in accordance to ClinGen TP53 Expert Panel Specifications : https://www.clinicalgenome.org/affiliation/50013 | |||||
18 | TP53 | Giacomelli et al, 2018 | 30224644 | ClinGen TP53 Expert Panel Specifications | ClinGen TP53 Expert Panel Specifications | ClinGen Expert group recommends combining Nutlin-3 and Etoposide assays to assess for variant dominant negative effect (DNE) and loss of function (LOF) Nutlin assay (DNE) = Nutlin-3 z-score of p53WT cells compared to z score for silent variants Etoposide assay (LOF) = Etoposide z-score of p53-null cells compared to z score for silent variants | p53WT nutlin-3 z-score <0.61 (>3SD from z score for silent variants) AND p53-null Etoposide z-score >-0.21 (>3SD from z score for silent variants) (i.e. no evidence of DNE + no evidence of LOF) | p53WT Nutlin-3 z-score ≥0.61 (>3SD from z score for silent variants) AND p53-null Etoposide z-score ≤-0.21 (>3SD from z score for silent variants) (i.e. evidence of DNE + LOF) | Supplementary Table 3 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical group. Use evidence weighting in accordance to ClinGen TP53 Expert Panel Specifications : https://www.clinicalgenome.org/affiliation/50013 | |||||
19 | MLH1/ MSH2 | Bouvet et al, 2019 | 30998989 | Strong | Strong | Mean survival score = average survival fraction (%) after 2 and 3 treatments with 0.1 μM MNNG. | Mean survival score <58.9% | Mean survival score >82.2% | 39 | 20 | 19 | 0.51 | 20 | 0 | 0 | 0 | 19 | 0.95 | 0.05 | 19.00 | 0.05 | One discordant result where clinical classification questioned removed from counts. | Clare Turnbull | ICR | ||||
20 | MLH1/ MSH2 | Drost et, 2019 | 30504929 | Strong | Strong | CIMRA assay (MMR activity) scores converted to Odds of Pathogenicity (which equates to a Likelihood Ratio) | OddsPath (LR) < 0.05 | OddsPath (LR) > 18.7 | Supplementary Table 2 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Odds Pathogenicity calculated using positive and negative controls. This equates to a likelihood Ratio (LR). If Odds Path (LR)>18.7, strong evidence towards pathogenicity (4 points) can be awarded. If Odds Path (LR)<0.05, strong evidence towards benignity (4 points) can be awarded. | |||||
21 | MSH2 | Jia et al, 2021 | 33357406 | Strong | Strong | PLEASE NOTE: Designation of functional status may be less robust for scores close to zero (both negative and positive) and therefore caution should be exercised in the application of these results under PS3/BS3. Data review aiming to inform more explicit recommendations is underway. | Loss of function (LOF) score = log2-ratio of variant’s frequency after 6-TG treatment divided by its frequency after mock treatment (6-TG is selectively toxic to MSH2 proficient cells). | LOF score <0 | LOF score > 0 | 63 | 42 | 21 | 0.67 | 41 | 0 | 0 | 1 | 21 | 0.98 | 0.09 | 20.50 | 0.05 | Two discordant results where clinical classifications questioned removed from counts. | Alice Garrett | ICR | Yes | ||
22 | PTEN | Mighell, Evans-Dutson & O'Roak, 2018 | 29706350 | Moderate (ClinGen PTEN VCEP v3.0) | Supporting (ClinGen PTEN VCEP v3.0) | Follow ruling from ClinGen PTEN VCEP guidelines v3.0 | Saturation mutageneis assay. "In the supplementary material (Table S2) search for the variant in columns A or B and make sure the variant in question is listed as TRUE under column I (high confidence). If not, do not use as evidence. Under column G, the cumulative score is listed. Apply PS3_moderate for all variants with scores < -1.11." | Cumulative score > 0 | Cumulative score <-1.11 | Table S2 | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | N/A | Assay recommended as suitable for use in variant classification by the relevant expert ClinGen clinical groups (PTEN VCEP). Misense variants with observations in gnomAD were considered Benign and variants reported as LP/P in ClinVar without conflicting reports considered as Pathogenic. | Sophie Allen | ICR | ||
23 | VHL | Buckley et al., 2024 | 38969834 | Moderate | Strong | Function score > -0.23 | Function score < -0.39 | Supplementary Table 1 | 57 | 42 | 15 | 0.74 | 36 | 0 | 6 | 1 | 14 | 0.97 | 0.13 | 12.86 | 19.6 | Charlie Rowlands | ICR | |||||
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